Chemoenzymatic Semisynthesis of Proteins

Robert E Thompson; Tom W Muir

doi:10.1021/acs.chemrev.9b00450

. Author manuscript; available in PMC: 2021 Mar 25.

Published in final edited form as: Chem Rev. 2019 Nov 27;120(6):3051–3126. doi: 10.1021/acs.chemrev.9b00450

Chemoenzymatic Semisynthesis of Proteins

Robert E Thompson ¹, Tom W Muir ^1,^*

PMCID: PMC7101271 NIHMSID: NIHMS1061594 PMID: 31774265

Abstract

Protein semisynthesis – defined herein as the assembly of a protein from a combination of synthetic and recombinant fragments - is a burgeoning field of chemical biology that has impacted many areas in the life sciences. In this review, we provide a comprehensive survey of this area. We begin by discussing the various chemical and enzymatic methods now available for the manufacture of custom proteins containing non-coded elements. This section begins with a discussion of methods that are more chemical in origin and ends with those that employ biocatalysts. We also illustrate the commonalities that exist between these seemingly disparate methods and show how this is allowing for the development of integrated chemoenzymatic methods. This methodology discussion provides the technical foundation for the second part of the review where we cover the great many biological problems that have now been addressed using these tools. Finally, we end the piece with a short discussion on the frontiers of the field and the opportunities available for the future.

1. INTRODUCTION

Nearly twenty years after the completion of the human genome project,¹ we have an ever-increasing understanding of the ~ 20,000 human genes and the roles of the proteins which they encode. At the time the catalogue was first announced, it represented a modest number of entries considering the ostensible complexity of the human organism. Today it is apparent that the total size of the human proteome vastly exceeds this genomic tally, a discrepancy explained by an explosion in the molecular diversity arising through spliced isoforms and post-translational modifications (PTMs). The nature of each of these unique proteoforms and how they contribute to cellular biology is the subject of extensive research,² one that increasingly relies on our ability to manipulate the chemical structure of this class of biomolecules both in vitro and in vivo.

The primary sequence of recombinant proteins may be modified through site-directed mutagenesis, genetically mutating a residue for any of the other 19-common proteinogenic amino acids. Mutagenesis of proteins has been used extensively to characterize structure–function relationships, and as a means to augment biological activity. A key challenge in the modification of protein structure, however, is the ability to introduce chemical functionality that is not normally genetically encoded. In doing so, the effects of protein dynamics, PTMs and protein-protein interactions may be laid bare through the introduction of biophysical probes, amino acid modifications and chemical crosslinkers respectively. The direct chemical labeling of reactive amino acid side-chains, such as cysteine, is widely used to modify proteins with various chemical functionalities.³ Alternatively, the ribosomal incorporation of unnatural amino acids using synthetic biology tools is a powerful way to install chemically unique side-chains into a protein.⁴ However, engineering of polypeptides using the tools of organic chemistry and chemical peptide synthesis provides the most selective and flexible means to make modified protein sequences, able to not only modify amino acid side chains, but to also alter the chemistry and chirality of the underlying peptide backbone.⁵ Merging the chemical flexibility of peptide synthesis with the power of recombinant protein engineering is therefore an incredibly attractive route to deciphering how protein structure dictates function.

Approaches to the synthesis of peptides and proteins have historically centered on the chemical condensation of amino acids and peptide precursors into longer polypeptide chains. The earliest such example by Curtius in 1882 is the chemical synthesis of the achiral diglycine molecule from acid chlorides and amines (Figure 1a).⁶ In the >130 years since, the condensation of amines and carboxylic acids remains the dominant strategy of amide bond synthesis, although it has been greatly refined. Indeed, a practitioner of medicinal or peptide chemistry has a choice of dozens of chemical ‘coupling reagents’ that facilitate the reaction of carboxylic acids with amines to yield amides in high yield.⁷ In conjunction with the development of protecting groups that mask the amino acid side-chains from the reactive coupling conditions,⁸ the ability to assemble peptides on a solid support through iterative coupling steps (Solid-Phase Peptide Synthesis – SPPS)⁹ makes it possible to chemically assemble polypeptides in a user-defined manner reminiscent of ribosomal synthesis (Figure 1b). Despite the flexibility and efficiency of contemporary peptide synthesis, it remains difficult to routinely prepare peptides longer than ~50 amino acids in length, far below the average size of globular protein domains.¹⁰ Accessing synthetic polypeptides and proteins beyond this length requires convergent assembly from smaller synthetic fragments. Condensation reactions between fully protected synthetic peptides may be carried out using peptide coupling reagents (i.e. classic fragment condensation¹¹), however such building blocks are notoriously difficult to manipulate in both aqueous and organic solvents, a property that limits this convergent synthetic approach.

Figure 1. — Pioneering examples of peptide bond construction through a) condensation between activated acyl donors and amines, b) Solid-Phase Peptide Synthesis (SPPS), and c) Native Chemical Ligation (NCL – see Figure 3 for additional mechanistic detail).

This problem of how to perform fragment condensation in an operationally simple manner prompted a shift in thought towards how to ligate purified unprotected peptides in aqueous solvent through selective amide bond synthesis.¹² This eventually led to the introduction in 1994 of the Native Chemical Ligation (NCL) method by Kent and coworkers who showed that peptides bearing C-terminal thioesters (α-thioesters) and N-terminal cysteine residues respectively can condense under aqueous conditions in high yield (Figure 1c).¹³ Due to the efficiency of NCL and extensions of this reaction, totally synthetic proteins well in excess of 100 amino acids containing all manner of backbone or side-chain chemical modification may be assembled through iterative ligations of synthetic peptides. Generally speaking, however, the size and number of chemical modifications that one might wish to install into a protein represents a minor proportion of its overall structure. For example, it might be desirable to change just a few residues in an enzyme active site, while leaving all other positions (i.e. most of the molecule) unmodified. Consequently, when synthesizing modified proteins through ligation chemistry, the majority of effort and resources are directed towards the stepwise chemical assembly of peptides containing the standard 20 amino acids. A semisynthetic approach to protein synthesis offers an expedient solution to this problem, wherein a protein is assembled from both synthetic and recombinant fragments, the latter derived from ribosomal protein synthesis, which is both cost effective and unburdened by protein-length limitations.

The synthesis of proteins requires chemoselectivity emblematic of bio-orthogonal chemistry: the ability to forge an amide bond selectively in the presence of the cloud of reactive functionalities commonly present within a polypeptide. This can be achieved by installing chemically orthogonal reactive handles during peptide chemical synthesis. Clearly, when dealing with recombinant proteins, this reactivity must be somehow genetically encoded. Chemical ligation methods that use the reactivity of proteinogenic amino acids, such as cysteine (for NCL) or serine/threonine (Serine/Threonine Ligation – STL),¹⁴ are amenable to such extension. However, the full implementation of the semisynthetic strategy requires the ability to install functionalities into recombinant proteins that are not directly incorporated by the ribosome. These include the high-energy α-thioester functionality needed for NCL. This turns out to be a less prosaic protein-engineering problem, one whose solution requires the repurposing of various biocatalytic facilitators. For example, a class of proteins known as inteins emerged as ideal tools for the installation of α-thioesters into recombinant proteins.¹⁵ In parallel, approaches for amide bond generation through enzymatic transpeptidation have also appeared, using modified protease¹⁶ or transpeptidase¹⁷ enzymes (Figure 2). Each of these biocatalysts promote biochemical transformations that are very similar to those that occur during NCL, cleaving and reassembling amide bonds through a series of N-to-S/O acyl shift reactions.¹⁸ The innate activity of these biocatalysts therefore makes them especially suited for the semisynthetic manipulation of proteins.

Figure 2. — Timeline of major developments in contemporary protein semisynthesis.

Contemporary protein semisynthesis resembles a manner of biomolecular-orthopedics. The polypeptide backbone, fittingly named, can be altered with high precision, introducing artificial groups throughout. In this review, we inspect each of the ways that enzymes have been used in the semisynthesis of proteins from synthetic and recombinant building blocks. In doing so we hope to harmonize these distinct methods, which share overlapping chemical transformations. We aim to highlight how this chemistry can be leveraged to forge backbone amide bonds on recombinant proteins, whose reactive functionalities are exposed and must be negotiated. In particular, we wish to highlight the power of more recently developed methods employing enzymes and autoprocessing split-intein domains that have been co-opted to serve as biorthogonal protein-based ‘coupling reagents’. We then describe how the protein semisynthesis strategy has been applied to the biochemical and biophysical analysis of proteins. Finally, we offer a perspective on the role these technologies have yet to play for hypothesis-driven biochemistry, particularly in the native cellular context. The total chemical synthesis of proteins (i.e. through the ligation of two or more polypeptides derived exclusively from chemical peptide synthesis), for which there are many excellent recent reviews,^{5, 19, 20} falls outside the scope of this monograph. Similarly, methods for bioconjugation using the innate reactivity of amino acid side-chains,^{3, 21} will not be discussed, nor will the remarkable advances that have been made in the last two decades allowing the ribosomal incorporation of many unnatural amino acids.^{4, 22}

2. CHEMICAL LIGATION OF EXPRESSED PROTEINS

Protein semisynthesis merges the fields of protein biotechnology and chemical peptide synthesis. The former area has seen remarkable advances thanks to the tools of molecular and synthetic biology, while the latter has been continually refined over the course of the century to the point of routine automation (at least for small to modest sized peptides). Thus, bringing these two powerful capabilities together in a single manifold offers an appealing route to manipulate the chemical structure of very large proteins. In a sense the best of both worlds can be achieved within a semisynthetic – i.e. part recombinant part chemical – protein framework. This tantalizing prospect, recognized for decades,^{23, 24} has fueled tremendous technological advances in the last two decades. In the following sections, we discuss how NCL, which is without question the dominant approach for the total chemical synthesis of proteins, has been successfully extended into the realm of protein semisynthesis. In so doing, we highlight how biocatalytic transformations have been instrumental in furnishing recombinant proteins with the necessary functionalities for chemical ligation.

2.1. Native Chemical Ligation

Native chemical ligation is a highly selective amide-bond forming reaction that has been used broadly for the total synthesis and semisynthesis of proteins. The reaction condenses two completely unprotected polypeptide fragments in neutral aqueous conditions, one reaction partner functionalized as an N-terminal cysteine (or reactive equivalent) and the other as an α-thioester (Figure 3). Peptide ligation is initiated by a dynamic trans-thioesterification reaction between the sulfhydryl group of the N-terminal cysteine and the α-thioester moiety, a process that has been generally observed between thiols and acyl-donors including thioesters,²⁵ the rate of which depends on the pKa values of incoming and outgoing nucleophiles. The resulting transthioesterified intermediate then spontaneously rearranges through an S-to-N acyl shift with the pendant α-amine through a 5-membered ring intermediate, creating an amide bond at the ligation junction. The reaction cascade central to NCL was first demonstrated in 1953 by Wieland and coworkers,²⁶ who successfully condensed cysteine and valinyl-thioester. It took over 40 years for this reaction to be applied to the ligation of synthetic polypeptides,^{13, 27} but then only two years subsequent to be extended to expressed proteins.²⁸

Figure 3. — The mechanism of native chemical ligation (NCL).

The chemistry underlying NCL capitalizes on the unique reactivity of the native cysteine residue. At physiological pH, the sulfhydryl functionality on the cysteine side-chain is in equilibrium with the nucleophilic thiolate form (pKa ~8.5).²⁹ In the cell, this potent nucleophile is utilized in the active sites of the eponymous class of cysteine protease enzymes.³⁰ Processes involving thioester formation and acyl migration are central to several biological processes, including protein ubiquitination,^{31, 32} transglutamination,³³ in the biosynthesis of non-ribosomal peptides,³⁴ intein splicing^{35, 36} and the covalent modification of bacterial cell-surface proteins.^{37, 38} (See sections 2.2: “Inteins and Expressed Protein Ligation”, 3.1 “Artificially split inteins”, and 4.1 “Sortase A” for details regarding these last two mechanisms and how they have been co-opted for biotechnology.) Clearly, the biocompatibility of the underlying chemistry of NCL implies the extension of this approach to biomolecules such as recombinant proteins – a capability that was noted in the initial disclosure of the method.¹³

The rate-determining step in NCL is the initial trans-thioesterification reaction. This bimolecular reaction is not templated, and therefore requires sufficient concentrations of reactants to outcompete hydrolysis of labile thioester reactants. Kent and coworkers investigated the importance of the nature of the α-thioesters used in the reaction, observing accelerated ligations in the presence of aryl thioesters.³⁹ Nonetheless, a reaction between a cysteinyl peptide and a preformed alanyl α-thiophenylester is significantly less rapid (second order rate constant of 0.26 M⁻¹s⁻¹)⁴⁰ than the commonly used thiol-maleimide bioconjugation reaction (second order rate constant ~300-800 M⁻¹s⁻¹).^{40, 41} The kinetics of the NCL reaction is also dependent on the nature of the C-terminal amino acid within the α-thioester fragment. Smaller amino side-chains afford more rapid reactions, whereas β-branched amino acids such as valine and isoleucine are more sluggish to react.⁴² Typical NCL reactions require therefore that at least one of the reactants be in millimolar concentration for efficient conversion to products.

2.1.1. Chemical synthesis of N-terminal cysteinyl peptides

Synthesis of N-terminal cysteinyl peptides for ligation reactions is routine in peptide chemistry, owing to the range of protecting groups available for masking this reactive side-chain.⁸ These reagents may be prepared by either the Boc or Fmoc-strategy for SPPS (Figure 4), where the sidechain sulfhydryl is protected from acylation, alkylation or oxidation by either benzyl (Bn), p-methylbenzyl (4-MeBn), p-methoxybenzyl (4-MeOBn) or triphenylmethyl (Trt) protecting groups respectively.^43–45 Deprotection of the sulfhydryl group then occurs concomitantly with the global deprotection and cleavage with either hydrogen fluoride (Boc-strategy) or concentrated trifluoroacetic acid (Fmoc-strategy) in the presence of carbocation-scavenging reagents. Alternatively, protecting groups orthogonal to the cleavage conditions, such as commonly used S-acetamidomethyl (Acm) and thiazolidine allow the cysteine sulfhydryl to remain protected after peptide purification.^{46, 47} This strategy, in addition to orthogonal protection of the cysteine α-amine,⁴⁸ has allowed for sequential multi-step peptide and protein assemblies.^{47, 49}

Figure 4. — Synthesis of N-terminal cysteinyl peptides using either Boc or Fmoc-strategy solid-phase peptide synthesis. (PG = protecting group).

2.1.2. Chemical synthesis of peptide α-thioesters

Compared to N-terminal cysteinyl peptides, peptide α-thioesters require a greater degree of synthetic manipulation to prepare, due to the relative instability of this functionality to basic conditions. (We direct the reader to the following reviews that focus on this topic.^{20, 50}) Through Boc-strategy SPPS, peptide α-thioesters may be synthesized directly on the solid support and remain stable to both the TFA and HF-based deprotection and cleavage conditions (Figure 5a).^{42, 51, 52} This strategy is incompatible with Fmoc-strategy SPPS due to the instability of thioesters to the repeated exposure to piperidine required for Fmoc-deblocking. Fully protected peptides synthesized using Fmoc-chemistry may be converted to α-thioesters under highly controlled conditions that minimize epimerization of the C-terminal amino acid.⁵³ Alternatively, methods that capitalize on late-stage activation of stable linkers have proven to be robust and flexible routes to α-thioesters (Figure 5b–d). An early example of this involved the use of an alkanesulfonamide “safety-catch” linker that could be activated for α-thioester installation following Fmoc-SPPS.⁵⁴ Comparable to the NCL reaction cascade in reverse, peptides bearing C-terminal 2-mercapto glycolate esters^{55, 56} and 2-mercapto alkylamides^57–59 can rearrange through acid-catalyzed O/N-to-S acyl shift to form α-thioesters that can be trapped by external thiols to form stable α-thioesters. Peptides functionalized with C-terminal diaminobenzoyl (Dbz) linkers may be converted to N-acyl ureas through on-resin acylation,^{60, 61} or to N-acyl benzotriazoles through treatment with acidic sodium nitrite solution,⁶² and subsequently thiolyzed with aryl or alkyl thiols. The use of N-acyl hydrazides, one of the first methods of acyl activation, has been reimagined as a thioester precursor for NCL reactions.^{63, 64} Chemoselective conversion of this comparatively stable moiety to an α-thioester can be carried out in situ prior to ligation reactions, a method that has been widely adopted for both protein total synthesis and semisynthesis.⁵⁰ Importantly, resins pre-functionalized with thioester precursors are now commercially available (including the Dbz, bis(2-sulfanylethyl)amino (SEA),⁵⁹ and N-acyl hydrazide linkers) significantly reducing the barrier to adoption of these powerful technologies.

Figure 5. — Common strategies for the synthesis of peptide thioesters by a) Boc and b-d) Fmoc strategy SPPS.

2.1.3. Expression of N-terminal cysteinyl proteins.

Accessing recombinant protein substrates for NCL has typically been achieved through expression of either full-length or truncated proteins in heterologous host organisms. As for all ribosomally synthesized proteins, genes encoding these proteins must contain an initiator methionine residue at its first position. Various methods, chemical and enzymatic, have been explored in order to transform these constructs into the necessary N-terminal Cys-containing proteins for NCL. The most direct method to install an N-terminal Cys into a recombinant protein is to rely on the endogenous methionine aminopeptidase to remove the initiator methionine from a precursor containing Met-Cys at the N-terminus (Figure 6).⁶⁵ The kinetics of this processing reaction is highly dependent on the nature of the second amino acid, the enzyme preferring smaller amino acids including Cys as well as Gly, Ala, Ser, Pro, Thr, Val.⁶⁶ After processing of the N-terminal Met residue, the free N-terminal Cys is prone to conversion in vivo to a thiazolidine through reaction with endogenous electrophilic metabolites such as pyruvate.⁶⁵ Regeneration of the N-terminal Cys must therefore be carried out prior to NCL, for example, through treatment with methoxylamine.⁶⁷ Hauser and Ryan employed a similar approach in their semisynthesis of a fusion lipoprotein, expressing the Cys-bearing protein of interested in E. coli as a fusion with the pelB leader peptide sequence (Figure 6).⁶⁸ This sequence directed the expressed protein to the periplasmic space where an endogenous signal peptidase liberated the N-terminal cysteine-containing protein from the fusion tag.

Figure 6. — Production of recombinant N-terminal cysteinyl proteins through *in vivo* proteolysis.

In vitro chemical treatment of proteins with cyanogen bromide has been widely used for the selective cleavage of amide bonds following methionine.⁶⁹ The reaction of this potent electrophile with the Met thioether side-chain generates an unstable iminolactone, which may be subsequently hydrolyzed (Figure 7). This method has been investigated as a chemical route to isolate fragments of erythropoietin for downstream ligation chemistry, requiring mutation of native methionine residues to avoid off-target cleavage.⁷⁰

Figure 7. — Mechanism for the chemical cleavage of Met-Cys bonds using cyanogen bromide.

The use of sequence specific proteases to liberate cysteinyl proteins in vitro provides an alternative to relying on the endogenous processing machinery of the cell (Figure 8a). The proteases Factor Xa^{28, 71, 72} and thrombin⁷³ have been widely used for this purpose due to their tolerance for cysteine at the P1’ positions of their recognition sequences. In the first reported use of NCL on recombinant proteins, Verdine and coworkers expressed the two members of AP-1 transcription factor complex (c-Jun and c-Fos) as N-terminal cysteinyl proteins, genetically fused to purification tags by a Factor Xa recognition sequence (Figure 8b).²⁸ Protease treatment liberated the N-terminal Cys proteins prior to NCL reactions with synthetic thioesters, in this case the small molecule chelator EDTA. Cleavage by both Factor Xa and thrombin requires careful optimization to avoid proteolysis of secondary sites. Such considerations motivated Tolbert and Wong to investigate the use of the highly specific Tobacco Etch Virus (TEV) protease as a way to liberate cysteinyl proteins.⁷⁴ Besides the increased sequence-specificity relative to Factor Xa and thrombin, this protease may also be overexpressed in E. coli, making this a more accessible and cost effective option.⁷⁵ Additionally, TEV protease remains active in low concentrations of additives such as detergents and denaturants, conditions that are favorable when working with otherwise insoluble protein fragments.⁷⁶

Figure 8. — Production of recombinant N-terminal cysteinyl proteins through proteolysis of fusion proteins *in vitro*. a) Commonly used proteases for the generation of N-terminal cysteinyl proteins for NCL. b) Preparation of recombinant cFos leucine zipper domain with an N-terminal cysteine for NCL (Erlanson et al.).²⁸ c) Preparation of recombinant N-terminal cysteinyl truncated H3 for the semisynthesis of H3K9me3 (Nguyen *et al.*).⁷⁷ Inset: Structure of the Smt3/Ulp1 complex (PDB ID code 1EUV).

The small ubiquitin-like modifier (SUMO) protein, often used to boost soluble protein expression, may be removed selectively from fusion proteins through treatment with specific SUMO proteases.⁷⁸ The Saccharomyces cerevisiae SUMO protein Smt3 and the corresponding protease Ulp1 are the most commonly employed pairing, although other combinations exist.⁷⁹ Unlike the proteases described previously, SUMO-proteases recognize both the tertiary fold of SUMO as well as the cleavage sequence at the C-terminus (Xaa-Gly-Gly|Xaa), providing highly specific proteolysis (Figure 8c). Like TEV protease, Ulp1 retains activity under mildly denaturing conditions that are often necessary for the purification of aggregation-prone proteins such as histones.⁸⁰ These features make the Smt3/Ulp1 system especially well-suited for the preparation of N-terminal Cys proteins for use in NCL. Indeed, this SUMO-fusion strategy has emerged as perhaps the ‘go to’ method for the generation of this class of ligation-ready recombinant building block.

2.1.4. Chemical routes to recombinant protein α-thioesters.

As discussed above, many methods for the preparation of synthetic peptide α-thioesters are available, relying on the ability to control chemical manipulations on specific carbonyl groups during peptide synthesis. Such precise control is facile in a chemically defined synthetic peptide, but exceedingly challenging on a recombinant polypeptide. Nonetheless, there has been some success in exploiting certain amino acid sequence motifs embedded within a protein precursor as a way to directly chemically install α-thioesters. For example, MacMillan and coworkers showed that C-terminal cysteines are prone to an N-to-S acyl shift rearrangement under acidic pH, a feature that has been exploited to generate α-thioesters.⁵⁷ The method has been adapted to afford recombinant protein-hydrazides, which were subsequently transformed into α-thioesters for use in protein ligation applications.^81–83 In an elegant example, Otaka and coworkers showed that Ni(II) ions promote the N-to-O acyl shift of Ser-Xaa-His motifs, generating peptide oxo-ester intermediates that can be converted to acyl hydrazides through treatment with hydrazine.⁸⁴ The related use of this Ni(II)-mediated N-O shift reaction for cleavable protein-tags suggests that this reaction could be extended to the preparation of recombinant protein hydrazides for use in NCL.⁸⁵

While these chemical cleavage strategies have utility in specific contexts, they do not offer a general solution to the problem of recombinant protein α-thioester generation. Fortunately, a biocatalytic solution to this problem has been developed and, interestingly, this also involves targeted cleavage of a specific peptide bond, albeit in this case facilitated by a remarkable family of auto-processing proteins known as inteins.

2.2. Inteins and Expressed Protein Ligation

Many proteins become processed through peptide bond cleavage as part of their natural maturation and biological regulation.⁸⁶ Typically this is achieved in trans through the action of dedicated protease enzymes that catalyze the hydrolysis of a specific amide bond in the precursor polypeptide. However, processing may also occur in cis through the action of auto-processing proteins. Discovered in 1990,⁸⁷ inteins are such a class of protein domain that catalyze a biochemical process known as protein splicing. These domains, genetically embedded in larger protein-coding genes, undergo splicing post-translationally, which involves the cleavage and re-ligation of the two peptide-bonds immediately flanking the intein domain (Figure 9). Thus, the appended protein domains flanking the intein, referred to as the N- and C-exteins, become ligated together without any traces of the excised intein. Once regarded as curiosities restricted to a small number of microorganisms, we now know, thanks to modern genomic and metagenomics efforts, that inteins are in fact widespread in nature, found in all three branches of life and in many viruses.^{88, 89} Indeed, these genomic efforts have revealed astonishing functional diversity within the intein family, and as we shall see in the coming sections, this has opened up tremendous opportunities in the semisynthesis area including, but certainly not restricted to, providing a straightforward solution to α-thioester installation in recombinant proteins.

Figure 9. — Post-translational modification of proteins through intein-mediated protein splicing.

2.2.1. Mechanism of intein-mediated protein splicing

The basic chemical mechanism of protein splicing was elucidated in 1993 by Perler and coworkers.⁹⁰ Subsequent in vitro biochemical and structural analyses by several research groups mean that we understand the process in great detail.⁹¹ Protein splicing follows a series of acyl-shift reactions highly reminiscent of transformations that occur in the NCL reaction cascade (Figure 10). In the canonical mechanism, the first residue of the intein domain (Cys or Ser) undergoes nucleophilic attack on the carbonyl of the preceding amide-bond which is activated by conserved catalytic residues. The resulting oxy(thio)ester linkage then undergoes reversible trans-(thio)esterification with the first residue of the C-extein (exclusively Cys, Ser or Thr) to form a branched oxy(thio)ester intermediate. Resolution of this intermediate then occurs through cyclization of the conserved C-terminal Asn residue of the intein to form a succinimide. This step liberates the intein domain and triggers a spontaneous O/S-to-N acyl shift rearrangement resulting in the N- and C-exteins being joined by a normal peptide bond. Divergence from this mechanism of protein splicing occurs in a small minority of inteins that lack a nucleophilic N-terminal residue, and that instead form the branched oxy(thio)ester intermediate directly.⁹² However, mutation of this highly conserved feature generally redirects the splicing pathway of the intein towards a cleavage byproduct, where cyclization of the C-terminal asparagine residue occurs prior to forming the branched intermediate, leading to autoproteolysis of the C-extein from the intein. Alternatively, mutation of the C-terminal asparagine of the intein can abolish the ability to undergo branched-intermediate resolution, causing a build-up of activated oxy(thio)ester which can hydrolyze to cleave the N-extein from the intein.⁹² This autoproteolytic activity makes inteins attractive tools in biotechnology as traceless protein purification tags.^93–95 Critically, exogenous thiols may partake in nucleophilic trapping of the acyl-intein thioester, thus liberating the N-extein as an α-thioester,⁹⁶ which can be isolated and subjected to chemical ligation reactions with synthetic (or recombinant) cysteinyl polypeptides. This semisynthetic extension of NCL using recombinant protein α-thioesters has come to be known as Expressed Protein Ligation (EPL),⁹⁷ or less commonly Intein-mediated Protein Ligation.⁹⁸

Figure 10. — Biochemical mechanism of protein splicing and associated N- and C-terminal cleavage pathways.

2.2.2. Production of recombinant protein α-thioesters using inteins.

The protein splicing activity of an intein is, to the first approximation, independent of the nature of the appended N- and C-exteins.⁹¹ This biochemical promiscuity is key to the generality of the EPL approach and accounts for the very wide range of systems that have been accessed using the technology. Foundational work in this field employed the vacuolar ATPase subunit intein from Saccharomyces cerevisiae (Sce VMA intein). Indeed, in one of the first applications of EPL, Muir, Sondhi and Cole used this intein in the semisynthesis of the 450-residue protein tyrosine kinase C-terminal Src kinase (Csk), a target beyond the synthetic limits of practical protein chemical synthesis (Figure 11).⁹⁷ Recombinantly expressed C-terminally truncated Csk, fused to the Sce VMA intein, was treated with synthetic peptides corresponding to the C-terminus of Csk in the presence of thiophenol, which served to liberate a reactive protein α-thioester from the intein. Similarly, Xu and coworkers found that the use of thiophenol and mercaptoethanesulfonate (MESNa) were effective thiol catalysts for both the liberation of protein α-thioesters from both the Sce VMA and Mycobacterium xenopi DNA gyrase A (Mxe GyrA) inteins, and subsequent ligations with synthetic peptides.⁹⁸ Notable, β-mercaptoethanol and dithiothreitol, while effective for promoting N-terminal extein cleavage, were found to be poor catalysts for the subsequent ligation reaction, presumably due to the known instability of thioesters derived from these reagents.^{95, 99} In addition to thiolysis, activated protein of interest (POI)-intein fusions can be cleaved by hydrazine to generate protein acyl-hydrazides.^{63, 100} Liu and coworkers found that cleavage of a fusion between microtubule-associated protein light chain 3 (LC3) and Mxe GyrA could be induced by hydrazine instead of the commonly used thiol MESNa.⁶³ Moreover, cleavage was more efficient with hydrazine and the hydrazide product is chemically more stable than the corresponding α-thioester. Considering the widespread adoption of synthetic peptide acyl-hydrazides as thioester precursors for ligation reactions, it is tempting to predict increased use of intein hydrazinolysis compared to thiolysis in future.

Figure 11. — Expressed protein ligation (EPL) for the semisynthesis of modified Csk.⁹⁷

Mxe GyrA has been the most widely used intein for the generation of recombinant protein α-thioesters (see Table 1 for a comprehensive listing of EPL applications). The reasons for this are partly historical and partly practical. This intein was among the first to be discovered and made available to the biomedical community.¹⁰¹ However, the small size of this intein relative to other inteins (198 aa), combined with its ability to be refolded from a denatured state, make it attractive when generating protein α-thioesters from insoluble or aggregation prone proteins.¹⁰² The Mxe GyrA intein has also been amenable to engineering to increase expression of fusion proteins.^103–105 Premature cleavage of the POI-GyrA fusion in vivo can be largely suppressed through a Thr-to-Cys mutation at position 3 of the intein, which temporarily inactivates the catalytic Cys1 residue through the formation of a disulfide.¹⁰⁵ Strömgaard and coworkers demonstrated that the GyrA sequence can be reduced by up to 25% through the replacement of superfluous structural elements with unstructured linkers, affording higher yields of protein but without compromising thiol-induced cleavage activity.¹⁰⁴ In addition to rational engineering efforts, Shusta and coworkers demonstrated that directed evolution of the Mxe GyrA intein can be used to afford more highly expressing POI-intein fusions (including antibody fragments).¹⁰³ These studies demonstrate the intrinsic malleability of certain inteins, in this case Mxe GyrA, in order to maximize the production of protein α-thioesters.

Table 1.

Applications of NCL and EPL to protein semisynthesis.^a

Protein	Location of synthetic modification	Modification
Abl tyrosine kinase	Central	Fluorophore labeling¹⁰⁶
	n/a	Segmental Isotopic Labeling¹⁰⁷
Adapter protein crk-II	n/a	Segmental isotopic labeling^{108, 109}
Akt1 serine-threonine kinase	C-terminal	Ser473Ph¹¹⁰, Ser477Ph¹¹⁰, Ser479Ph¹¹⁰
β-Amyloid	C-terminal	Unmodified¹¹¹
Androgenic Gland Hormone	C-terminal	N-linked glycosylation¹¹²
Anthrax Lethal Factor	N-terminal	N-terminal acetylation¹¹³
Apolipoprotein E (ApoE)	n/a	Segmental Isotopic Labeling¹¹⁴
Arylalkylamine N-acetyltransferase (AANAT)	N-terminal	Thr31Ph, ¹¹⁵ Thr31Pma¹¹⁶
	C-terminal	Ser205Ph,^{115, 117} Ser205Pfa^{115, 117}
Aspartate transporter Glt_Ph	C-terminal	Arg397Citrulline¹¹⁸
Autophagy-related protein 3 (Atg3)	N-terminal	Lys19Ac¹¹⁹
	Central	Lys48Ac¹¹⁹
Azurin	C-terminal	Cys112Sec¹²⁰, Met121Nle¹²¹, Met121Sec¹²², Met121Sem¹²¹, Met121Hcy¹²³
Casein kinase II (CK2)	C-terminal	Thr344Ph¹²⁴, Ser347GlcNAc¹²⁴
Chemotaxis protein CheA	n/a	Segmental Isotopic Labeling¹²⁵
Checkpoint kinase 2 (CHK2)	N-terminal	Thr68Ph¹²⁶
Chorismate mutase	C-terminal	Arg90Cit,¹²⁷ βArg9¹²⁸
Chaperone protein ClpB	n/a	Segmental isotopic labeling¹²⁹
	N-/C-terminal	Fluorophore labeling¹³⁰
Chaperone protein DnaK	n/a	Segmental Isotopic Labeling¹³¹
Chemokine (C-X-C motif) ligand 8 (CXCL8)	C-terminal	C-terminal truncation¹³², fluorescein¹³³, benzoylphenylalanine¹³⁴, β-peptide mimetics¹³⁵, segmental isotopic labeling¹³⁶
Chemokine (C-X-C motif) ligand 12 (CXCL12)	C-terminal	6-Nitroveratryl (Nvoc) photocages^{137, 138}, fluorescein¹³⁹
γD-Crystallin	n/a	Segmental isotopic labeling¹⁴⁰
5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase	n/a	Unmodified¹⁴¹
Eph receptor tyrosine kinase	C-terminal	Unmodified^{142, 143}
Erythropoietin (EPO)	N-terminal	N-linked oligosaccharide,^{144, 145} alkyne tag¹⁴⁶
Eukaryotic initiation factor-4A (elF4A)	C-terminal	Ser209Ph¹⁴⁷
Glutamyl-prolyl-tRNA synthetase	n/a	Segmental Isotopic Labeling¹⁴⁸
Glutaredoxin	N-terminal	Cys11Sec¹⁴⁹
Glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1)	N-terminal	Thr/SerGalNAc¹⁵⁰
	C-terminal	Thr/SerGalNAc¹⁵⁰
Glycogen synthase	C-terminal	Thr668Ph¹⁵¹
α-Hemolysin	Central	Thr117Pra¹⁵², Thr117LysAcac¹⁵³
Heterogeneous nuclear ribonucleoprotein L (hnRNPL)	n/a	Segmental isotopic labeling¹⁵⁴
Histone H2A	N-terminal	Lys5Ac¹⁵⁵, Lys9Ac¹⁵⁵, Lys13Ac¹⁵⁵, Lys13Ub⁸¹, Lys15Ac¹⁵⁵, Lys15Ub⁸¹, Lys15Ub (H2A.X)¹⁵⁶
	C-terminal	Ser139ph (H2A.X)¹⁵⁶, Lys118Ac¹⁵⁵ Lys119Ub^{157,81, 158}
Histone H2B	N-terminal	Ser14ph,⁶⁷ Lys5Ac,⁶⁷ Lys11Ac,⁶⁷ Lys12Ac,⁶⁷ Lys15Ac⁶⁷, Lys16Ac¹⁵⁵, Lys20Ac¹⁵⁵, Lys34Ub⁸¹
	C-terminal	Lys108Ac¹⁵⁵, Ser112GlcNAc¹⁵⁵, Lys116Ac¹⁵⁵, Lys120Ac¹⁵⁵, Lys120Ub¹⁵⁹, Lys120Su¹⁶⁰, Lys125Ac¹⁵⁵
Histone H3	N-terminal	Arg2me2¹⁵⁵, Lys4Ac¹⁶¹, Lys4me3^162–164, Gln5Sero¹⁶⁵, Lys9me3^{161–164, 166–169}, Lys9Ac^{77, 161}, Lys14Ac^{77, 161}, Lys18Ac^{77, 161, 170}, Lys18Cr¹⁷¹, Lys23Ac^{161, 170}, Lys27me3^{162–164, 172}, Lys23Ac¹⁷⁰, Lys36me3^{155, 164}
	Central	Lys79me2/3¹⁶⁴, Tyr41Ph¹⁵⁵, Arg42me2¹⁷³
	C-terminal	Lys115Ac¹⁷⁴, Lys122Ac¹⁷⁴
Histone H4	N-terminal	Arg3me2¹⁵⁵, Lys5Ac,^{77, 161, 170} Lys5Cr¹⁷¹, Lys8Ac^{77, 161, 170}, Lys8Cr¹⁷¹, Lys12Ac^{77, 161, 175} Lys12Cr¹⁷¹, Lys12Su¹⁷⁶, Lys16Cr¹⁷¹, Lys16Ac,^{77, 175, 177} His18pTza¹⁷⁸, Lys20Ac,⁷⁷ Lys20Cr¹⁷¹, Lys20me3¹⁶⁸
	C-terminal	Lys77Ac¹⁵⁵, Lys79Ac¹⁵⁵
Heterochromatin protein 1α (HP1α)	C-terminal	Atto532 labeling¹⁷⁹,¹⁶⁶ fusion with shigoshin peptide¹⁷⁹
Heat shock protein 27 (Hsp27)	C-terminal	Arg188Apy¹⁸⁰
Heat shock protein 90 (Hsp90)	n/a	Unmodified¹⁸¹
Huntingtin	N-terminal	Thr3Ph^{182, 183}, Lys6Ac¹⁸³, Ser13Ph¹⁸⁴, Ser16Ph¹⁸⁴
Immunity protein Im7	N-terminal	N-linked glycosylation¹⁸⁵
Interferon Response Factor 3 (IRF3)	n/a	Segmental isotop[ic labeling¹⁸⁶
Interleukin 6	Central	N-linked glycosylation¹⁸⁷
Interleukin 13	Central	N-linked glycosylation¹⁸⁸
Jarid2	N-terminal	Lys116me3^{189, 190}, Ser120Ph¹⁹⁰, Ser124Ph¹⁹⁰, Ser126Ph¹⁹⁰
KcsA potassium channel	C-terminal	Unmodified^{102, 191}, Gly77d-Ala^192–194, Gly79ester¹⁹³
	Central	Trp68Bta¹⁹⁵, Val76ester¹⁹⁶, Val76(¹³C¹⁸O)¹⁹⁷, Gly77ester,¹⁹⁶ Gly77(¹³C¹⁸O)¹⁹⁷, Gly79ester,¹⁹⁶ Gly79(¹³C¹⁸O)¹⁹⁷, Gly77d-Ala^{193, 196}
KH-type splicing regulatory protein	n/a	Segmental Isotopic Labeling¹⁹⁸
KvAP potassium channel	Central	G198dA,¹⁹²
Lacticin 481 precursor (LctA)	C-terminal	Unmodified¹⁹⁹, sequence variants²⁰⁰, Thr33Ph^{201, 202}, Cys38Sec²⁰³, Asn39Hse²⁰⁴, Asn39Nva²⁰⁴, Asn39Cba²⁰⁴, Asn39dAsn²⁰⁴, Met40Pra²⁰⁴, Met40Nle²⁰⁴, Met40Nva²⁰⁴, Met40dMet²⁰⁴, Asn41Cba²⁰⁴, Trp43Nal²⁰⁴, Ser28dCys²⁰⁴, Gly29Sar²⁰⁴, Ile31Sar²⁰⁴, His32Sar²⁰⁴, Ile34Sar²⁰⁴, Glu37Sar²⁰⁴
Low molecular weight protein tyrosine phosphatase (LML-PTP)	C-terminal	Tyr131Pmp,²⁰⁵ Tyr132Pmp²⁰⁵
Microcin B17 precursor (McbA)	C-terminal	Sequence variants²⁰⁶, Cys41Hcy
Microtubule-associated protein light chain 3 (LC3)	C-terminal	Unmodified⁶³, lipidation^207–210
Mitogen-inducible gene 6 (Mig6)	C-terminal	Tyr394Ph²¹¹
Mxe GyrA intein	N-terminal	Isotopic labeling²¹²
	C-terminal	Isotopic labeling²¹³, Branched isopeptide intermediate,²¹⁴ Val182hVal²¹⁴, Thr⁺¹Dap^{213, 214}, His187ThA^{213, 214}, Asn198Nva²¹³
NaK ion channel	C-terminal	Asp66Csa,²¹⁵ Asp66Hse²¹⁵
NEDD4-like E3 ubiquitin-protein ligase WWP2	N-terminal	Tyr369Ph²¹⁶
Neurosecretory protein GM	C-terminal	Unmodified²¹⁷
Notch (V1711 – α-secretase cleavage product)	N-terminal	N_α-Ac²¹⁸
Nucleolar protein 3 (NPL3)	n/a	Segmental isotopic labeling¹⁵⁴
Npu DnaE split intein	n/a	Segmental isotopic labeling²¹⁹
cGMP phosphodiesterase	C-terminal	Crosslinker (BPA)²²⁰
p300 HAT domain (WT and circular permutation)	C-terminal	Unmodified,²²¹ Lys1546Ac,²²² Lys1549Ac,²²² Lys1551Ac,²²² Lys1554Ac,²²² Lys1558Ac,²²² Lys1560Ac,²²²
p38α MAP kinase	C-terminal	Thr323Ph²²³
Paxillin	N-terminal	Tyr31Ph,²²⁴ Tyr31Ph(caged)²²⁴
Peroxiredoxin-1	C-terminal	Lys197Ac²²⁵
Phosphatase and tensin homolog (PTEN)	C-terminal	Ser380Ph,^226–228 Thr382Ph, ^226–228 Thr383Ph,^226–228 Thr385Ph,^226–228
Postsynaptic density protein 95 (PSD-95)	N-terminal	Backbone ester mutants^{229, 230}, Ser73Phos (PDZ1)²³¹
	C-terminal	Tyr236Ph (PDZ2)²³¹, Tyr240Ph (PDZ2)²³¹, Y397ph (PDZ3)²³¹
Polypyrimidine tract binding protein	n/a	Segmental Isotopic Labeling^{148, 232}
Prion Protein	C-terminal	Unmodified,²³³ GPI anchor^{234, 235}, Palmitoyl anchor^{234, 236}, PEGylation,²³⁷
Prochlorosin precursor (ProcA)	C-terminal	[²H]Ser13²³⁸
Protein Kinase A	C-terminal	ATP-conjugate²³⁹
Rab7 GTPase	C-terminal	Fluorophore labeling²⁴⁰, Cys(Ger)-OMe^241–244, diprenylation²⁴²
Rad53 serine-threonine kinase	N-terminal	Thr5Ph,²⁴⁵ Thr8Ph,²⁴⁵ Thr12Ph,²⁴⁵ Thr15Ph²⁴⁵
H-Ras GTPase	Central	Gln61GluOMe²⁴⁶, palmitoylation²⁴⁷
K-Ras4B GTPase	C-terminal	Cys(Ger)-OMe^248–250, Ser181Ph²⁴⁸
Rheb GTPase	C-terminal	Cys(Ger)-OMe²⁴⁹
Ribonuclease A (RNase A)	C-terminal	Unmodified⁹⁸, Cys110Sec²⁵¹, β-turn mimics^252–256, N-linked glycosylation^{257, 258}
Ribonucleotide reductase (RNR)	C-terminal	Tyr365Ni²⁵⁹, fluorinated Tyr365²⁶⁰, Tyr365Dopa²⁶¹
S-Adenosylhomocysteine hydrolase	C-terminal	Lys401Ac²⁶², Lys408Ac²⁶²
Selenoprotein M (SelM)	n/a	Unmodified (Sec-containing)²⁶³
Akt1 serine-threonine kinase	C-terminal	Ser473Ph¹¹⁰, Ser477Ph¹¹⁰, Ser479Ph¹¹⁰

SHP-1 phosphatase	C-terminal	Tyr536Pmp/Pfp²⁶⁴, Tyr580Pmp/Pfp²⁶⁴
SHP-2 phosphatase	C-terminal	Tyr542Ph,²⁶⁵ Tyr542Pmp/Pfp,²⁶⁵ Tyr580Ph,²⁶⁵ Tyr580Pmp/Pfp,^{265, 266}
Smad2	C-terminal	Ser465Ph^{267, 268}, Ser467Ph^{267, 268}, diazirine photocrosslinker²⁶⁹, photocages^270–272
Sortase A	C-terminal	Cys184Sec²⁷³, Cys184Hcy²⁷³, Arg197Cit²⁷⁴
Src tyrosine kinase	C-terminal	ATP-conjugate²⁷⁵, Tyr527(2-MeTyr)²⁷⁶, Tyr527(b-MeTyr)²⁷⁶, Tyr527Hty²⁷⁶, Tyr527(2,6-F₂Tyr)²⁷⁶
	Central	Lys57Orn²⁷⁷, Lys57Dab²⁷⁷, Lys57Dap²⁷⁷
Signal Transducer And Activator Of Transcription 6 (STAT6)	C-terminal	Tyr641Ph (+photocage)²⁷⁸
α-Synuclein	N-terminal	Backbone thioamide^{279, 280}, Nα-Ac²⁸¹, Lys6Ub²⁸² Lys12Ub₄²⁸³
	C-terminal	Tyr125Ph²⁸⁴, Tyr125Ni²⁸⁵, Ser129Ph^{286, 287}
	Central	Tyr39Ph²⁸⁸, Thr65GlcNAc²⁸⁹, Thr72GlcNAc^{289, 290}, Thr75GlcNAc²⁹⁰, Ser81GlcNAc²⁹⁰, Ser87GlcNAc^289–291
	N-/C-terminal	Fluorophore labeling²⁹²
Tau protein	C-terminal	Unmodified²⁹³, Ser396Ph⁴⁹, Ser400GlcNAc²⁹⁴, Ser404Ph^{49, 295}, Ser422Ph²⁹⁶,
	Central	Lys280Ac⁴⁹, Lys294Cm²⁹⁷, Ser293Ph²⁹⁷, Ser305Ph²⁹⁷, Tyr310Ph⁴⁹,
Thioredoxin reductase	C-Terminal	Unmodified (Sec-containing)²⁹⁸, Cys-to-Hcy and -Sec mutants^299–305
Thymidine monophosphate kinase	N-terminal	Lys25NBD-Dap³⁰⁶
Transforming growth factor beta (TGFβ) receptor 1	C-terminal	Thr185Ph,^{307, 308} Ser187Ph,^{307, 308} Ser189Ph,^{307, 308} Ser191Ph^{307, 308}
Trypsin	N-terminal	Unmodified³⁰⁹
Tyrosine-protein kinase Csk	C-terminal	TyrPh⁹⁷
Ubiquitin^b	C-terminal	Diazirine crosslinker³¹⁰, K6Ub⁸³, K48Ub³¹¹, Ser65Ph³¹², non-hydrolyzable AMP-ester mimic^{313, 314}
Uracil DNA glycosylase	N-terminal	Thr6Ph³¹⁵, Tyr8Ph³¹⁵
Zif268 Zinc finger transcription factor	C-terminal	Arg70IDAO,³¹⁶ Arg70Pra,³¹⁶ His85TACN,³¹⁶ Lys89Fl³¹⁶
β-Xylanase	N-terminal	Fluorinated Glu78³¹⁷
Yeast α-factor mating receptor	n/a	Segmental Isotopic Labeling³¹⁸
Ypt1 GTPase	C-terminal	prenylation^{319, 320}
Zinc finger protein (QNK-QDK-RHR)	C-terminal	Arg78Cit³²¹

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Ac, acetyl; Acac, acetatoacetyl; Apy, argpyrimidine; Bta, β-(3-benzothienyl)-alanine; Cba, cyano β-alanine, Cit, citrulline; Cm, carboxymethyl; Csa, cysteine sulfonic acid; Dab, diaminobutyrate; Dap, diaminopropionate; Fl, fluorescein; GalNAc, N-acetylgalactosaminyl; Ger, geranyl; GlcNAc, N-acetylglucosaminyl; Hcy, homocysteine; Hse, homoserine; Hty, homotyrosine; hVal, hydroxylvaleric acid; IDAO, iminodiacetic acid ornithine; Nal, napthyl alanine; NBD-Dap, N₃-nitrobenzofurazane-l-1,3-diaminopropionic acid; Ni, nitro; Nle, norleucine; Nva, norvaline; Orn, ornithine; Pfa, phosphonodifluoromethylenealanine; Pfp, phosphonomethylenephenylalanine; Pmp, phosphonomethylenephenylalanine; Pra, propargylglycine; Ph, phosphoryl; Pma, phosphonomethylenealanine; pTza, phosphoryltriazoylalanine; Sar, sarcosine; Sec, selenocysteine; Sem, selenomethionine; Sero, serotonyl; Su, sumoyl; TACN, triazacyclononane; ThA, 2-thienylalanine; Ub, ubiquityl.

Including the assembly of protein sequences from recombinant and synthetic peptide and protein fragments. Does not include the use of NCL/EPL as a general bioconjugation strategy, e.g. for the generation of antibody conjugates.

Semisynthesis of the protein sequence of ubiquitin itself. Examples of semisyntheses of ubiquitin-modified proteins are listed under the protein of interest.

2.3. Extensions and Alternatives to Native Chemical Ligation

The only strict requirement for a potential native chemical ligation junction within a protein target is that it contains a cysteine residue. While this might not seem like too much of a constraint, the low abundance of cysteine within the proteome (1.1%)³²² makes the chances of a suitably located native ligation junction actually quite low. Of course, one can simply introduce a non-native cysteine residue into the target through either mutation or insertion. This commonly employed solution does come with attendant risks given the nucleophilicity and redox activity of the sulfhydryl group. This problem has fueled a great deal of effort geared to towards expanding the number of potential disconnection sites for protein total- and semi-synthesis (Figure 12).

Figure 12. — Extending ligation sites beyond Cys through a) Ser/Thr ligation, b) NCL followed by Cys alkylation, c) NCL followed by desulfurization (not limited to examples of β-thiol amino acids shown – we direct the reader to the following reviews^{323, 324}), d) traceless-NCL using removable ligation auxiliaries,²⁰ e) NCL at selenocysteine followed by selective deselenization (not limited to examples shown – we direct the reader to the following reviews^{324, 325}).

Chemical ligation may be performed between a peptide bearing an N-terminal serine or threonine and a peptide α-ester derived from a suitable hydroxy aldehyde, such as glycolaldehyde or salicaldehyde.^{326, 327} Selective condensation between the two functionalities occurs through reversible oxazolidine formation, which then rearranges through an O-to-N acyl shift of the proximal ester to form the ligated product (Figure 12a). Salicaldehyde ester auxiliaries can then be removed through acidolytic treatment, generating a native peptide. By virtue of the abundance of serine and threonine residues in the proteome (6.6% and 5.5% respectively)³²² the use of Ser/Thr ligation (STL) in a semisynthetic context offers many potential ligation junctions to disconnect the target. In a study by Kirshenbaum and coworkers, the STL reaction was extended from the ligation of exclusively synthetic constructs to the recombinant polypeptides Ribonuclease S-protein and parathyroid hormone (1-34).³²⁸ Recombinant proteins bearing N-terminal serines can be obtained through proteolytic liberation strategies akin to those discussed above for NCL, however installation of the required α-ester group into a recombinant fragment is theoretically less straightforward, somewhat limiting the generality of the STL strategy in the semisynthesis arena.

The structure and function of many proteins is unaffected by the introduction of unnatural cysteine residues for the purposes of NCL/EPL reactions (Table 1). However, for proteins that are not amenable to such alteration, there have been considerable methodological developments that expand the approach beyond this rare amino acid. The simplest of these strategies involves elaborating the sulfhydryl group of the cysteine post-ligation (Figure 12b). For example, alkylation can be used to form a range of thioether-containing mimics of proteinogenic side-chains, including lysine,^{165, 329} glutamine,³³⁰ and glutamate.³³¹ Notably, this strategy does not discriminate between the cysteine at the ligation junction and any other cysteines present in the protein. The use of homocysteine as a ligation partner also provides access to methionine residues through methylation after the ligation reaction.^{332, 333} While not one of the standard 20-amino acids, this residue in protected form may be ribosomally incorporated into recombinant proteins through unnatural amino acid incorporation.³³⁴ Alternatively, Petersson and coworkers demonstrated that disulfide-protected homocysteine can be enzymatically installed on the N-terminus of proteins through the activity of a bacterial aminoacyl transferase, a method the authors used for a semisynthesis of α-synuclein.³³⁵

Cysteine residues may be formally mutated to alanine through chemical desulfurization reactions (Figure 12c), which expands potential ligation junctions to this highly abundant amino acid.^{336, 337} As for the use of alkylation reactions post-ligation, such methods are incompatible with endogenous cysteine residues, which will also be chemically modified. As a consequence, these strategies have been extensively applied to the semisynthesis of cysteine-free proteins, such as ubiquitin, α-synuclein and histones (histone H3 contains a single conserved cysteine that may be mutated to alanine) (Table 1). Alternatively, non-participating cysteine residues in recombinant proteins may be masked selectively, e.g with phenacyl groups,¹⁸⁰ which can then be unmasked after the ligation and desulfurization reactions. To expand to further ligation junctions, derivatives of proteinogenic amino acids bearing sufhydryls at the β- or γ-position have been synthesized and incorporated into synthetic peptides for use in ligation reactions.^{323, 324} These hybrid amino acids partake in ligation reactions with synthetic or recombinant polypeptide α-thioesters through the NCL/EPL reaction pathway and may be subsequently converted to the native residue through desulfurization. Of particular note is the use of β-mercapto aspartate by Becker and coworkers in the semisynthesis of pegylated forms of full-length prion protein (PrP).²³⁷ The perturbed electronics of β-mercapto aspartate³³⁸ allowed this residue to be selectively desulfurized in the presence of two native cysteine residues that are necessary for stabilization of the protein through disulfide formation. Thiolated lysine residues have also been used in the synthesis and semisynthesis of ubiquitinated proteins.^339–341 In this case, the non-native sulfydryl reacts with synthetic or recombinant ubiquitin (or potentially SUMO) α-thioesters and direct acyl-shift to the to the ε−amine of Lys to form an isopeptide linkage. An important extension of this strategy is the ability to incorporate δ-mercapto lysine site-specifically into recombinant proteins through unnatural amino acid incorporation,²⁷⁷ work that improved upon an earlier, related approach involving incorporation of cysteinyl-ε-lysine into recombinant proteins.³⁴² An alternative to using non-native amino acids is instead the use of traceless acyl-transfer auxiliaries appended directly to the reactive amine, bearing appropriately positioned sulfhydryl groups (Figure 12d).³⁴³ Such auxiliaries are much more sensitive to the steric load surrounding the ligation junction and have been used most effectively at unhindered ligation junctions (e.g. Gly-Gly), for example in the semisynthesis of ubiquitinated proteins.^{81, 344, 345}

Native chemical ligation can also be extended to cysteine’s chalcogen cousin, selenocysteine.^{251, 346, 347} The selenol group of selenocysteine is substantially more acidic than that of cysteine, existing predominantly as the nucleophilic selenolate at physiological pH, although oxidation to the corresponding diselenide or mixed selenylsulfides is highly facile.^{325, 348} Akin to the NCL pathway, trans-thio/selenoesterification with polypeptide α-thioesters generates the corresponding Se-linked intermediate that subsequently rearranges through an Se-to-N acyl shift. Raines and coworkers demonstrated that this chemistry is amenable for use in protein semisynthesis, through the reaction with a recombinant α-thioester fragment of ribonuclease A generated through thiolysis of a Mxe GyrA intein fusion.²⁵¹ Although a genetically encoded amino acid, selenocysteine is not easily introduced into recombinant proteins, which has limited the use of recombinant N-terminal selenocysteinyl proteins as ligation partners. Rozovsky and coworkers were able to express selenocysteine-containing recombinant proteins for ligation through supplementation of the growth medium with selenocystine, leading to the misloading of the cysteinyl-tRNA.²⁶³ The resulting selenocysteine-containing proteins were liberated using TEV protease to generate the N-terminal selenocysteinyl protein for ligation. Notably, this strategy will also result in replacement of any internal cysteines in the recombinant fragment with selenocysteine. Thus, in cases where site-specific selenocysteine incorporation is desirable, other approaches are necessary, including unnatural amino acid incorporation^349–351 or enzymatic installation using aminoacyl transferase enzymes.³⁵²

Initially used as a way to prepare natural selenoproteins, the use of selenocysteine in protein semisynthesis has recently been directed towards the selective chemistry that may be conducted at this amino acid. In particular, selenocysteine can be converted to alanine or serine through phosphine-mediated deselenization under reductive or oxidative conditions, respectively.^{353, 354} Importantly, reductive selenocysteine-to-alanine conversion may be carried out in the presence of cysteine residues (Figure 12e), offering an alternative to ligation-desulfurization for proteins containing endogenous cysteine residues. In line with this approach, selenated derivatives of proteinogenic amino acids have been prepared for use in ligation-deselenization chemistry.³²⁴ Additionally, use of α-selenoesters have been used as acyl-donors that are more reactive than the corresponding α-thioesters with both N-terminal cysteinyl and selenocysteinyl peptides,^{355, 356} allowing peptide ligations at junctions that may be otherwise intractable (e.g. C-terminal proline).³⁵⁷ While this class of reagents has yet to be extended to protein semisynthesis, this would seem to be a highly productive line of enquiry for the future. Analogous to the generation of α-thioesters, the use of inteins bearing a catalytic selenocysteine residue could provide access to protein α-selenoesters through trans-selenoesterification to extend this chemistry to expressed proteins.³⁵⁰

3. PROTEIN TRANS-SPLICING

The discovery and characterization of protein splicing in cis by contiguous inteins was a clear demonstration that the acyl-shift chemistry utilized in protein ligation chemistry also occurs in nature to alter the primary structure of proteins. However, the development and discovery of trans-splicing split inteins cemented the portrayal of these proteins as nature’s protein ligases. In this case, the fragmented intein pieces associate before auto-catalyzing protein splicing between two separate polypeptides (Figure 13). As one or both of these coupling partners can be synthetic, split inteins offer a chemoenzymatic approach for semisynthetic protein engineering. In this section we will examine the development of protein trans-splicing applications.

Figure 13. — Protein trans-splicing (PTS) mediated by split inteins.

3.1. Artificially split inteins

Many contiguous inteins contain a large homing endonuclease domain (HED) embedded within their sequence (Figure 14a). The presence of the HED is thought to allow the horizontal spread of inteins (along with the embedded HED) into host genomes.³⁵⁸ However, the HED is functionally separate from the protein splicing activity of the intein, and there are several examples where this insert has been genetically removed to generate fully active cis-splicing mini-inteins.^{359, 360} A remarkable feature of HED-containing inteins is the ability of the surrounding protein splicing element to assume its topologically intertwined fold, despite having a very large protein disrupting its primary sequence (Figure 14b). This suggested that it might be possible to reconstitute the intein fold in trans, i.e. by complementation of two separate polypeptide fragments (herein referred to as Int^N and Int^C) that would normally flank the HED. Indeed, in 1998 it was independently demonstrated in several studies that functional inteins could be reconstituted by refolding N- and C- fragments of the protein (Figure 15a).^361–364 Moreover, it was quickly realized that the protein trans-splicing (PTS) phenomenon could be used as a protein ligase system, through trans-splicing between an expressed protein Int^N fusion and a synthetic Int^C peptide.³⁶⁵

Figure 15. — Strategies for converting contiguous inteins into trans-splicing split inteins through a) refolding,^361–364 b) induced-proximity,³⁶⁶ and c) reconstitution under native conditions.³⁷¹

Several other contiguous inteins have been artificially split at the conserved HED insertion site within their sequence (Table 2). In most cases, a refolding step is required to obtain PTS activity, analogous to the Mtu RecA intein. However, there are some exceptions to this rule. For example, the artificially split Sce VMA intein can undergo PTS without a refolding step if (and only if) the two fragments, VMA^N and VMA^C, are brought into proximity using a two- or three-hybrid paradigm (Figure 15b).^366–370 In another example, Mootz and coworkers demonstrated that the artificially split Ssp DnaB intein can effect PTS under native conditions, albeit with reduced efficiency compared to the parental contiguous intein (Figure 15c).³⁷¹

Table 2.

Artificially and naturally split inteins.

Intein name	Type	k_splice (s⁻¹)^a	t_1/2^b	Fragment lengths^c (Int^N/Int^C)	Reference
Sce VMA	Artificially split	1.2 x 10⁻³	10 min	184/64	^{366, 371}
Ssp GyrB	Artificially split	6.9 x 10⁻⁵	170 min	150/6	³⁷³
Ssp DnaB	Artificially split	9.9 x 10⁻⁴	12 min	104/47	³⁷¹
Ssp DnaB	Artificially split	4.0 x 10⁻⁵	290 min	11/142	^{377, 378}
Ssp DnaB (M86)	Artificially split	2.5 x 10⁻³	5 min	11/142	³⁷⁸
Ssp DnaX	Artificially split	1.9 x 10⁻⁴	60 min	144/6	³⁷⁵
Ssp DnaE	Naturally split	6.6 x 10⁻⁵	175 min	123/35	^{379, 380}
Npu DnaE	Naturally split	3.7 x 10⁻²	19 s	102/35	^{380, 381}
Ava DnaE	Naturally split	3.1 x 10⁻²	23 s	102/35	³⁸⁰
Cra DnaE	Naturally split	1.2 x 10⁻²	58 s	118/35	³⁸⁰
Csp DnaE	Naturally split	1.8 x 10⁻²	39 s	99/35	³⁸⁰
Cwa DnaE	Naturally split	5.0 x 10⁻³	140 s	106/35	³⁸⁰
Mcht DnaE	Naturally split	2.4 x 10⁻²	29 s	104/35	³⁸⁰
Oli DnaE	Naturally split	1.6 x 10⁻²	43 s	112/35	³⁸⁰
Ter DnaE	Naturally split	8.5 x 10⁻³	82 s	101/35	³⁸⁰
gp-41–1	Naturally split	1.8 x 10⁻¹	4 s	88/36	³⁸²
gp41–8	Naturally split	4.5 x 10⁻²	15 s	91/44	³⁸²
NrdJ-1	Naturally split	9.8 x 10⁻²	7 s	105/39	³⁸²
IMPDH-1	Naturally split	8.7 x 10⁻²	8 s	101/41	³⁸²
AceL TerL	Naturally split	1.7 x 10⁻³	7 min	25/104	³⁸³
GOS TerL	Naturally split	1.1 x 10⁻²	65 s	37/152	^{384, 385}

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In vitro splicing rates under optimal conditions.

Calculated from reported first order rate constants.

Not including extein or initiator methionine residues.

Beyond splitting a contiguous intein at the canonical HED insertion site, considerable effort has been made towards finding alternative split sites with the goal of reducing the size of one of the intein fragments such that it becomes more accessible to SPPS (Table 2, Figure 16).³⁷² The canonical intein split site affords a large Int^N fragment (~90-100 aa) and shorter Int^C (~30-40 aa) – the former is well beyond the reach to standard SPPS, while latter is close to the limit of synthetic accessibility, thereby allowing only short C-extein cargoes to be appended. With this in mind, the Ssp GyrB mini-intein was artificially split close to the C-terminal splice junction, affording a 6-residue Int^C fragment that splices to ~80% completion (albeit with a 10-fold excess of the complementary Int^N component).³⁷³ The short Int^C sequence is easily accessible to peptide synthesis, and was subsequently used for C-terminal labeling of recombinant proteins.^{374, 375} Notably, this split site has been extended to other systems, specifically the Ssp DnaX and the Ter DnaE-3 mini-inteins.³⁷⁵ Alternatively, artificially split inteins can be generated through fragmenting the primary sequence proximal to the N-terminus, creating a short, synthetically accessible Int^N fragment (Figure 16). Accordingly, Mootz and coworkers showed that the Ssp DnaB intein could be artificially split to generate a short Int^N (11 aa), which was used to N-terminally label recombinant proteins in vitro.³⁷⁶ This split site was extended to the Rma DnaB intein, which was able to undergo moderately fast PTS (reaction half-life of 22 min) with a small Int^N (12 aa) affording >80% spliced product.³⁷⁵ Remarkably, Liu and coworkers demonstrated that PTS can occur in vivo with a DnaB intein split into 3 fragments, comprising a 49 aa C-terminal fragment, an 11 aa N-terminal fragment and a central 94 aa fragment.³⁷⁷

Figure 16. — Common sites for splitting inteins. a) Commonly explored sites for splitting the intein domain in the Int^N (S1, S2, S3) and Int^C (S10, S11) domains as well as the location of the endonuclease domain (EN/S0). β-strands (β1-β12) are indicated by grey arrows. Split site numbered according to Sun et al.³⁷⁷ b) Split sites mapped onto the structure of the *Ssp* DnaB mini-intein (PBD ID: 1MI8). The canonical Int^N and Int^C subdomains (*i.e.* the S0 split site) are colored light brown and orange respectively.

3.2. Naturally split inteins

In 1998 the first naturally split intein was discovered within the DnaE protein from the Synechocystis sp. strain of cyanobacteria (Figure 17).³⁷⁹ This split intein, named Ssp DnaE, consists of a 123-aa Int^N fragment and a 36-aa Int^C fragment (including initiator methionine), and likely evolved from a contiguous intein that underwent genomic rearrangement.³⁸⁶ In vitro characterization of this split intein revealed slow protein trans-splicing activity (first-order rate constant of 6.6 (± 1.3) x 10⁻⁵ s⁻¹),³⁸⁷ despite the two fragments associating extremely rapidly with nanomolar affinity (K_d < 50 nM).³⁶⁸ A homologue of Ssp DnaE from the Nostoc punctiforme (Npu) cyanobacterium was subsequently cloned and exhibited highly efficient splicing activity in vivo,³⁸¹ with greater tolerance to local extein sequence variation compared with Ssp. In vitro, Npu DnaE undergoes remarkably fast protein trans-splicing, exhibiting a reaction half-life of less than a minute, compared with > 60 min for Ssp DnaE.³⁸⁸ Further characterization of other DnaE split intein homologues revealed that ultra-fast splicing kinetics are common to this large family of split inteins.³⁸⁰ Indeed, the explosion of genomic sequencing data over the last decade has led to the identification of numerous naturally split inteins, many of which have been shown to have remarkable PTS activities. As a case in point, Mootz and coworkers characterized four non-allelic split inteins discovered through bioinformatic analysis of metagenomic sequence data.^{382, 389} These inteins (named gp41-1, gp41-8, NrdJ-1 and IMPDH-1) exhibited ultra-fast splicing kinetics, reacting to near-completion with half-lives less than 20s at low micromolar concentration.³⁸² Interestingly, these split inteins share only 40-50% sequence identity with Npu DnaE, supporting the notion that ultrafast splicing kinetics may be a conserved evolutionary feature amongst the majority of allellic and non-allellic split inteins. (In this regard, it is rather ironic that the first naturally split intein characterized, Ssp DnaE, supports comparatively slow PTS kinetics.) Indeed, biochemical analyses suggest that these unrelated splicing elements share a common folding mechanism,³⁹⁰ whereby strong electrostatic interactions drive the association of the split intein fragments, followed by an disorder-to-order “collapse” into the topologically intertwined intein fold to carry out protein splicing.²¹⁹ It should be noted, however, that the Npu DnaE, gp41-1, gp41-8, NrdJ-1 and IMPDH-1 split intein pairs do not cross-react, i.e. they are functionally orthogonal.³⁹⁰ This attractive feature allows for multiplexing-type applications.³⁹⁰

The discovery of naturally split inteins with atypical split sites further reinforces nature’s broad use of the PTS paradigm.^{383, 384} Two of these split inteins, both embedded in the large subunit of the T4-bacteriophage-type DNA-packaging terminase (TerL), originate from the environmentally disparate Ace Lake (AceL) in Antarctica, and the Punta Cormorant lagoon in the Galapagos (GOS). Both AceL-TerL and GOS-TerL have large Int^C fragments (104 aa and 152 aa, respectively), and correspondingly short Int^N fragments (25 aa and 37 aa, respectively) that are within the realm of synthetic accessibility. Interestingly, the AceL TerL split intein was most active at low temperatures (around 90% splicing yield with a half-life of ~7 minutes at 8 °C)³⁸³ while GOS TerL was most active at higher temperatures (around 90% yield with a half-life of between 1-3 min at 30 °C)^{384, 385} reflecting the evolutionary pressures imparted on both inteins. Two other recently discovered natural atypically split inteins, VidaL T4Lh-1 and VidaL UvsX-2, possess Int^N fragments that are even shorter than the TerL split inteins at 15-aa and 16-aa, respectively,³⁹¹ although the biochemical activity of these systems has yet to be fully explored. In contrast to the canonically split inteins, atypically split inteins appear to associate through hydrophobically driven association,³⁹² followed by disorder-to-order transitions upon complexation. Notably, while the folding mechanisms of canonically and atypically split inteins differ in detail, in both cases the pathways appear to have evolved such that autocatalytic side-reactions are avoided prior to fragment association, a problem that is often observed in artificially split inteins.³⁷²

3.3. Engineering of split inteins

While many naturally split inteins have highly favorable biochemical activities, at least relative to their artificially split intein counterparts, there is always room for improvement, whether it be increasing thermal stability or relieving functional constraints imposed by sequence restrictions at the splice junctions. Consequently, split inteins have been the focus of several protein engineering initiatives that have led to enhanced biochemical properties. Consensus protein engineering, wherein multiple sequence alignments are used to design an optimized sequence,³⁹³ has been used to improve the chemical and thermal stability of split inteins for PTS applications.^{392, 394} The application of this strategy to the DnaE family of split inteins, using 73 members predicted to possess fast-splicing kinetics,³⁹⁴ afforded a consensus-fast (Cfa) DnaE intein that possesses superior physicochemical properties and splicing kinetics relative to Npu DnaE, with which it shares 82% sequence homology (Figure 18). Strikingly, the splicing efficiency of Cfa DnaE was largely unaffected up to 8 M urea. The consensus design strategy was also applied to the atypically split TerL inteins, affording an consensus atypically split intein (Cat) with increased activity and stability,³⁹² suggesting that inteins are particularly amenable to this protein engineering strategy.

Figure 18. — Consensus designed DnaE split intein (Cfa DnaE).³⁹⁴ a) Sequence alignment of *Npu* DnaE and Cfa DnaE split intein. Differences are shown in grey. b) Sequence differences (blue sticks) mapped onto the structure of Npu (PDB ID: 4KL5).

The efficiency of PTS is somewhat context dependent and, as a general rule, all inteins show optimal splicing when the extein sequences at the immediate splice junctions (typically the first 2 or 3 positions of the N- and C-exteins) mimic those of the protein in which they are naturally embedded (see figure 14a for conventional numbering of extein residues common to cis- and trans-splicing inteins). The specifics and stringency of this dependency vary from case to case. For example, the Npu DnaE split intein requires a bulky aromatic residue at the +2 position of the C-extein; structural and functional studies have shown this residue stabilizes nearby catalytic residues in the intein.³⁹⁵ This so-called extein dependence has important implications for the use of split-inteins as tools for traceless protein semisynthesis, where splicing is required to occur in non-native and potentially unfavorable sequence contexts. Strategies employing directed evolution^{378, 396} and structure-based targeted mutagenesis³⁹⁷ have both been used successfully to relieve this extein dependence. Using PTS to assemble a kanamycin resistance gene in vivo provided a way to select for mutants of a DnaE split intein able to accept the -SGV- sequence at the Int-C-extein junction instead of the native -CFN-.³⁹⁶ This mutant intein was subsequently used to assemble the native sequence of the multidomain Crk-II protein. Of the four mutations responsible for this increased promiscuity, one substitution within a loop proximal to a catalytic His residue was itself capable of stimulating PTS by Npu DnaE when an unfavorable +2 Ala extein residue is present.³⁹⁵ Targeted engineering of this loop through saturation mutagenesis, again coupled to a cell-based selection system, generated an intein with a greatly relieved extein dependence that was subsequently used for the semisynthesis of a modified form of histone H3 (H3K27me3) in isolated nuclei.³⁹⁷ Liu, Mootz and coworkers similarly used directed evolution of the artificially split Ssp DnaB intein to promote more promiscuous splicing.³⁷⁸ Through sequential insertion of the contiguous intein into 10 different locations (with different local extein sequences) within the kanamycin resistance gene, the authors were able to select for inteins with an improved tolerance for non-native exteins. The split form of the best clone (“M86”) exhibited increased fragment affinity relative to the original split intein, as well as greater efficiency in PTS with both native and non-native exteins. It is important to consider that throughout known cis and trans-splicing inteins, each of the 20 common proteinaceous amino acids is sampled at both −1 and +2 extein positions.³⁹⁸

The discovery and engineering efforts described above are providing the protein engineer with an ever-expanding repertoire of split intein tools. From a practical perspective, these reagents are already able to catalyze protein trans-splicing at rates at or beyond the requirements of most protein semisynthesis applications, at least in vitro. Additionally, non-homologous inteins react orthogonally to one another, as the acyl-shift chemistry central to PTS is templated by the intein structure. This allows for multiple PTS reactions to occur simultaneously on the same^{368, 399} or separate³⁹⁰ polypeptides. The bio-orthogonality of the PTS reaction also makes it especially well suited to semisynthesis applications in living cells, in this case through the cytosolic delivery of synthetic peptides into mammalian cells expressing the complementary split intein fusion.^400–404 Notably, this same bio-orthogonality makes PTS an incredibly powerful tool in a host of other biomedical areas, including gene therapy^{405, 406} and synthetic biology.⁴⁰⁷ These exciting applications, as well as the many others discussed elsewhere in this review, will surely benefit from the continued discovery and subsequent engineering of split inteins with optimized properties tailored toward the system in question.

4. TRANSPEPTIDATION

Transpeptidation is the transformation of one peptide bond into another through the transacylation of amines. Enzymes that catalyze this reaction, known as transpeptidases, are used in nature to alter the primary sequence of proteins. These enzymes recognize and cleave specific motifs of amino acids, transferring the peptide acyl group to a suitable amine acceptor. In this section we will discuss the discovery and engineering of two families of transpeptidases, sortases and peptide asparaginyl ligases, which have been used extensively by the protein engineering community.

4.1. Sortase A

Gram-positive bacteria select a range of proteins for display on their cell surface through covalent attachment to the cell wall envelope.⁴⁰⁸ In S. aureus, each of these proteins destined for display contain a pentapeptide ‘sorting’ motif, LPxTG (x is variable). In 1999, a mutational screen in S. aureus identified a transpeptidase enzyme, a 206-residue protein containing a single cysteine residue, responsible for recognition of the sorting motif.⁴⁰⁹ This family of enzymes, dubbed sortases, is present in other gram-positive bacteria including B. subtilis, E. faecalis, S. pnuemoniae and S. pyogenes,⁴¹⁰ and most bacterial species contain multiple sortase enzymes (A through F), performing different roles in vivo, such as pilus polymerization. While the number of characterized and hypothetical sortase enzymes continues to grow,⁴¹¹ the most widely studied member of the family remains Sortase A from S. aureus (SaSrtA).

The enzymatic reaction carried out by sortases, and in particular Sortase A, has been widely studied in vitro and is well understood.^{412, 413} The ~145 residue transpeptidase domain of Sortase A (lacking the N-terminal signal peptide/membrane anchoring sequence) recognizes substrates containing the LPxTG sorting-motif (Figure 19a). The enzyme cleaves the amide bond between the Thr and Gly residues through the catalytic activity of the conserved nucleophilic cysteine, with base catalysis provided by a nearby histidine residue, generating an acyl-enzyme thioester intermediate. In the presence of glycine-terminated peptide nucleophiles, this intermediate is resolved through nucleophilic attack of the α-amine, resulting in a transpeptidation product. In vivo, this nucleophile corresponds to the oligoglycine chain in peptidoglycan cross-bridges. In vitro, peptide substrates containing a minimum of one N-terminal Gly residue are substrates for transpeptidation, although two or more glycines are preferred by the enzyme.⁴¹² The specificity for the pentapeptide motif has been illuminated through biochemical and structural analysis of the recombinant enzyme. Kinetic analysis of SaSrtA activity on peptide libraries demonstrated that only changes to position three of the LPxTG motif are tolerated by the enzyme.⁴¹⁴ An NMR structure of a covalent complex of SaSrtA and a substrate peptide mimic illuminated how this specificity of SaSrtA for the LPxTG sorting sequence is achieved, specifically through binding to a hydrophobic pocket adjacent to the active site (Figure 19b).⁴¹⁵

Figure 19. — Transpeptidation by sortase A. a) Catalytic cycle of SrtA-mediated transpeptidation. b) Close up of the SrtA-substrate complex (PDB ID: 2KID). Substrate is depicted in magenta.

The minimal substrate requirements of SaSrtA suggested that the catalytic activity of this enzyme could be used for enzymatic ligation of peptides and proteins. In a pioneering study, Mao and coworkers used the enzyme to conjugate a range of synthetic peptides to the C-terminus of the green fluorescent protein (GFP), including peptides containing modified amino acids.¹⁷ This method of protein conjugation, known as Sortase-Mediated Ligation (SML) and more simply “sortagging”,⁴¹⁶ offered a way to modify recombinantly expressed proteins in a controlled manner (Figure 20). The substrate requirements for a protein are minimal, namely that the protein contains either the short pentapeptide motif embedded within an accessible region of the protein (for C-terminal modification), or a N-terminal glycyl-acceptor sequence for (N-terminal modification). These substrates can be easily accessed through recombinant protein expression,⁴¹⁷ or through the action of SaSrtA itself.⁴¹⁸ Furthermore, cognate synthetic peptide substrates can be accessed through routine SPPS using standard amino acids. The simplicity of the reaction has led to sortagging being widely used to label proteins with a synthetic cargo.^{419, 420}

Figure 20. — Sortase-mediated ligation (sortagging) for the a) C-terminal or b) N-terminal modification of proteins of interest (POI).

Sortase-mediated transpeptidation reactions are inherently reversible, as the product (unavoidably containing a regenerated LPxTG motif) and the glycyl-leaving group are both substrates for the enzyme. In order to drive the reaction to completion, a large excess of the synthetic peptide is typically used to ensure that the reaction equilibrium is pushed towards the product side.¹⁷ As this is not always feasible (for example, the peptide may have limited solubility or be difficult to prepare in large amounts), several ways have been found to push the reaction towards completion. This has been achieved most simply through the removal of the small glycyl-leaving groups through dialysis (Figure 21a).^421–423 Alternatively, the introduction of a stable β-hairpin structure within the LPxTG motif, through the use of a tryptophan zipper, was found to increase the sortase-mediated ligation between two proteins (MBP and Trx), presumably forcing the sorting sequence in the product into a conformation that is not recognized by the enzyme (Figure 21b).⁴²⁴ Strategies that replace the pro-scissile Thr-Gly amide bond with a methyl ester⁴²⁵ or depsipeptide⁴²⁶ have been shown to increase the efficiency of the reaction, as these substrates generate methanol or hydroxylacetyl leaving groups that are not substrates for the reverse reaction (Figure 21c). Similarly, deactivation of the glycyl-leaving group through diketopiperazine formation,⁴²⁷ or through chelation in a nickel complex⁴²⁸ can prevent the leaving group from re-entering the catalytic cycle (Figure 21d,e). The use of metal chelation by Antos and coworkers is particularly convenient as it relies on native amino acids and can thus be used for peptide- or protein-bound sortase recognition motifs.⁴²⁸ In a conceptually distinct approach, Pentelute and coworkers found that by carrying the reaction out in a flow-based system, the concentration of acyl-acceptor could be decreased by an order of magnitude (300 μM in batch 10-20 μM in flow) while achieving comparable yields and fewer byproducts.⁴²⁹

Figure 21. — Strategies for improving the yield of sortagging reactions.

The low turnover rates of SaSrtA have motivated the engineering and evolution of this enzyme in order to increase the efficiency of transpeptidation (Figure 22). In an elegant application of directed protein evolution, Liu and coworkers used yeast display to anchor randomly mutated SaSrtA proteins to the cell surface, in proximity to one of the transpeptidation substrates.⁴³⁰ Iterative rounds of selection in the presence of decreasing concentrations of the cognate substrate afforded mutant sortase enzymes with up to 140-fold increase in catalytic efficiency compared with the wild type enzyme. Mutations to a loop flanking the LPxTG binding groove, conferred a ~30 fold reduction in Km for the sorting sequence.⁴³⁰ One of these mutants served as a starting point for additional improvements by Chen and coworkers, who used high throughput screening in vitro to identify additional beneficial mutations for enzymatic activity.⁴³¹

Figure 22. — Evolving the transpeptidase activity of sortase A. a) Schematic for the evolution of sortase activity using yeast display.⁴³¹ b) Locations of activating mutations mapped onto the structure of WT sortase A (PDB ID: 2KID). LPAT substrate shown in purple.

Using directed protein evolution, SaSrtA has also been reprogrammed to recognize sorting sequences other than the canonical -LPxTG- sequence.⁴³² This has important implications for protein semisynthesis applications, where the -LPxTG- scar in the product may affect the structure and function of the protein. Using their previously developed yeast display method for sortase evolution, Liu and coworkers were able to identify SaSrtA mutants, “eSrtA(4S)” and “eSrtA(2A)”, that recognize -LPxSG- and -LAxTG- sequences in preference to LPXTG.⁴³² The authors then demonstrate that the eSrtA(4S) enzyme is able to modify the native sequence of the protein fetuin A in human plasma. Schwarzer and coworkers used phage display to isolate SaSrtA mutants with relaxed substrate preference at the first position of the sorting motif.⁴³³ One such mutant (“F40”) preferred Ala, Asp, Ser, Pro and Gly over the native Leu, albeit at the expense of lower activity relative to the WT enzyme. However, screening of a second-generation library led to the identification of mutants with significantly improved activity for APXTG and FPXTG sequences.⁴³⁴ Importantly, the F40 sortase has found utility for the semisynthesis of N-terminally modified histone H3 through ligation at the native APATG sequence.^435–437

The transpeptidation activity of SaSrtA requires Ca²⁺, which stabilizes the structure of the enzyme.⁴¹⁵ The generation of Ca²⁺-independent mutants is of interest in order to extend the use of the enzyme inside living cells where Ca²⁺ levels are highly regulated. Sortase A from S. pyogenes (SpSrtA) is Ca²⁺-independent and has been used by Ploegh and coworkers for intra and intermolecular ligations in the cytosol of S. cerevisiae and HEK293T cells.⁴³⁸ Comparison of the Sortase A sequences from S. aureus and S. pyogenes revealed that calcium binding residues in SaSrtA can be mutated to the corresponding residues of SpSrtA (E105K, E108A/Q) to relieve Ca²⁺-dependence, although this does lead to a reduction in overall catalytic activity.⁴³⁹ However, combining these mutations with known activating mutations created SaSrtA variants with both increased activity and calcium independence.^440–442 These engineered sortases have been shown to be active in living cells, allowing for protein labeling in E. coli⁴⁴³ and 293T cells,⁴⁴⁴ and even in C. elegans.⁴⁴⁵ In a particularly impressive recent application, Lang and coworkers used a Ca²⁺-independent version of a re-programmed eSrt2A enzyme to ligate ubiquitin and SUMO to target proteins in live cells.⁴⁴⁴ Ribosomal incorporation of unnatural lysine residues, bearing isopeptide-linked diglycines as acyl-acceptors, allowed the generation of SUMOylated and ubiquitinated proteins in a site-specific manner, catalyzed by a calcium-independent, sequence-reprogrammed sortase (Figure 23).

Figure 23. — Sortase-mediated ubiquitination.⁴⁴⁴

Despite the widespread use of sortase-mediated transpeptidation for the conjugation of biochemical and biophysical probes to proteins,^{420, 446} its use in traceless protein semisynthesis has been limited compared to NCL/EPL. This is likely due to the sortagging ‘scars’ left behind by the process, which place a significant constraint on the design of traceless (or near traceless) semisynthetic schemes. Nonetheless, the continued use of sophisticated protein evolution methods to these enzymes suggests that these constraints will continue to be loosened, allowing sortagging to be increasingly employed in this area.

4.2. Peptide Asparaginyl Ligases

In plants, the formation of the cyclotide family of cyclic peptide natural products relies on enzymatic cyclization of the peptide backbone. This process is carried out by peptide asparaginyl ligase (PAL) enzymes,⁴⁴⁷ which may be considered a subset of ligase-type asparaginyl endopeptidases (AEP).⁴⁴⁸ Whereas AEPs primarily proteolyze Asx-Xaa bonds, PALs catalyze the transpeptidation of Asx-Xaa bonds to form intra- or intermolecularly ligated products. Two such PALs, butelase 1 and OaAEP1b, have been explored for use as peptide and protein transpeptidation catalysts.

4.2.1. Butelase 1

Tam and coworkers isolated a PAL enzyme from the butterfly pea C. ternatea that they named butelase 1.⁴⁴⁹ Unlike other asparaginyl endopeptidase enzymes, butelase 1 displays a preference for transpeptidation rather than hydrolysis of Asx-containing substrates (Figure 24), suggesting evolutionary specialization as a cyclase or ligase. Indeed, the ~38 kDa active enzyme was able to cyclize many non-native targets including kalata B1, SFTI, histatin-3 and thanatin in yields typically greater than 90% in a 1:400 ratio of enzyme-to-substrate. Importantly, butelase 1 was also able to catalyze transpeptidation in trans, between two small model peptides.

Figure 24. — Transpeptidation by butelase 1. a) Catalytic cycle of butelase 1-mediated transpeptidation. b) Close up of the butelase 1 active site complex (PDB ID: 6DHI).

Compared to sortase A, butelase 1 exhibits greater promiscuity in the sequences it is able to process. Biochemically, the enzyme processes the tripeptide motif –NHV- C-terminally to the asparagine residue, through the activity of a Cys-His-Asn catalytic triad (Figure 24b). The acyl-enzyme intermediate generated from this amidolytic reaction is then resolved through inter/intramolecular attack from an acyl-acceptor. Interestingly, attempts to intercept the acyl-enzyme intermediate with non-peptidic nucleophiles including thiols and hydrazine only afford products of hydrolysis instead of the corresponding thioester or hydrazide products.⁴⁴⁹ In an intermolecular context, a broad range of amino acids can be accepted as the first residue of the incoming acyl acceptor (i.e. the N-terminus), excluding acidic residues and proline.⁴⁴⁹ However, the enzyme has a strong preference for hydrophobic amino acids (Ile/Leu/Val) or Cys at the second position of the acyl acceptor peptide. In the case of intramolecular attack, specifically cyclization, these rules are much looser, and the enzyme is able to accept all amino acids at both the first and second positions, at least in certain combinations. Specifically, if either the first residue is Gly or the second residue is Leu, the adjacent position can be any of the 20 common amino acids. Surprisingly this sequence promiscuity even extends to many d-amino acids,⁴⁵⁰ which was used for the synthesis of analogues of cyclic peptide natural products consisting of d-amino acids except for the Asn-residue at the ligation site.

As per the reversibility of all known transpeptidation processes, butelase-mediated reactions liberate His-Val dipeptides that can serve as a competitive acyl acceptor, requiring excess substrate to drive the reaction to completion.⁴⁴⁹ To circumvent this problem, Tam and coworkers investigated the use of depsipeptide and thiodepsipeptide substrates for butelase 1.⁴⁵¹ The thiodepsipeptide substrates, releasing thiols unable to compete in the transpeptidation reaction, afforded ligations between peptide substrates in >90% yield after 1 h (Figure 25). This strategy allowed for the N-terminal labeling of recombinant GFP and ubiquitin proteins, as well as the synthesis of complex antimicrobial peptide dendrimers.⁴⁵² In the case of ubiquitin, where the accessibility of the N-terminus was limiting, adding a short linker afforded a labeling yield of ~95% with 0.001 equiv. of enzyme and only four equivalents of thiodepsipeptide reactant.

Figure 25. — Butelase 1-mediated labeling of ubiquitin using a thiodepsipeptide substrate.⁴⁵¹

4.2.2. OaAEP1b

OaAEP1b is a PAL enzyme that was isolated from the Oldenlandia affinis plant,⁴⁵³ the source of the cyclotide kalata B1.⁴⁵⁴ This enzyme can be expressed in E. coli (this is currently not possible for butelase 1) and is able to catalyze the cyclization of non-cyclotide polypeptide precursors,⁴⁵³ as well as the ligation of protein fragments.⁴⁵⁵ Like butelase 1, OaAEP1b recognizes and processes an -NXX- motif, where XX is typically ‘GL’ (the processed sequence within kalata B1), although ‘AL’ and ‘CL’ sequences considerably improved protein cyclization.⁴⁵⁵ The catalytic efficiency of this enzyme (~200 M⁻¹s⁻¹) is significantly lower than that of butelase 1 (~100,000 M⁻¹s⁻¹).⁴⁵⁶ Analysis of a crystal structure of recombinant OaAEP1b identified a Cys residue performing a gate-keeper role near the substrate channel of the enzyme, impeding the catalytic cyclization and ligase activity of the enzyme.⁴⁵⁶ Mutation of this residue to Ala afforded a PAL enzyme with significantly increased catalytic efficiency (~35 000 M⁻¹s⁻¹) for peptide cyclization, and that was able to catalyze efficient transpeptidation of ubiquitin to short synthetic peptides and to proteins.

Additional identification and characterization of PAL enzymes such as OaAEP1b and butelase 1 will continue to shed light on the factors that favor transpeptidation over hydrolytic activity, including the effects of so-called gate-keeper residues.^{447, 457} Such information will be critical for the further development of this fascinating class of enzymes, through directed evolution or rational design efforts, for a host of protein chemistry applications.

5. PEPTIDE LIGASES

It is well established that protease enzymes can catalyze amide bond formation through reverse proteolysis between amines and carboxylic acids under certain conditions. In order to capitalize on this activity, extensive efforts have been made to engineer proteases to carry out efficient peptide ligation reactions for the purposes of protein engineering, including semisynthesis. In this section we will describe how proteases have thus been converted into so-called ‘peptide ligases’.

5.1. Subtiligase

In one of the first examples of protein semisynthesis, the serine protease subtilisin BPN’ was used to reassemble RNase A from a complex of two proteolyzed fragments.⁴⁵⁸ This thermodynamically unfavorable reaction is promoted by the presence of organic co-solvents. To control reverse proteolysis reactions kinetically, activated ester substrates have been used as substrates in the place of C-terminal carboxylic acids.^{459, 460} In such cases, the preference for hydrolysis of acyl-enzyme intermediates in both cases is greater than the subsequent aminolysis by a suitable acyl acceptor. To avoid such back reactivity, Kaiser and coworkers explored enzymatic coupling of acyl-esters with thiolsubtilisin,⁴⁶¹ a chemically “damaged” mutant of subtilisin containing a Ser-to-Cys mutation in the active site (Figure 26a). ^{462, 463} Thiolsubtilisin, and indeed selenolsubtilisin,⁴⁶⁴ have greater preference for aminolysis over hydrolysis of acyl-enzyme intermediates, however it should noted that the initial esterase activity of the variants is below that of the WT enzyme.

Figure 26. — Peptide ligation using subtilisin variants. a) Synthesis of short peptides using thiolsubtilisin.⁴⁶¹ b) Total synthesis of ribonuclease A containing unnatural catalytic residues using subtiligase.⁴⁶⁵

In order to improve the catalytic activity of thiolsubtilisin, Wells and coworkers found that an additional mutation to the variant (P225A) was able to relieve steric crowing in the active site, improving the esterase efficiency of the enzyme, which they named ‘subtiligase’.⁴⁶⁶ In a landmark example of protein synthesis, Wells and coworkers used subtiligase to assemble ribonuclease A from seven synthetic fragments, incorporating unnatural fluorohistidine residues in the active site (Figure 26b).⁴⁶⁵ Acyl-donor peptides were suitably functionalized with C-terminal glycolate-phenylalanyl amide esters as leaving groups for the ligation reactions, which averaged 70%. Five additional mutations made to subtiligase afforded a variant named “stabiligase” that retained nearly 50% activity in 4M guanidine hydrochloride.¹⁶ This allowed enzyme-mediated ligation of a synthetic peptide glycolate ester to a recombinant version of human growth hormone (hGH) protein in the presence of denaturants.

Like the protease from which it is derived, subtiligase engages substrates in an extended conformation, recognizing four amino acids N-terminal to the cleavage/ligation site, and three residues to the C-terminal side.⁴⁶⁷ Unlike transpeptidase enzymes such as sortase A, subtiligase displays flexibility in the sequences that it can process in the P and P’ sites. On the N-terminal side of the ligation site, the P4 and P1 positions dictate much of the substrate specificity, preferring large hydrophobic residues.^{466, 468} At the P1’ and P2’ sites, C-terminal to the ligation site, subtiligase accepts most residues except for acidic or β-branched residues at the P1’ position.¹⁶ Weeks and Wells recently used a mass-spectrometry-based approach to further profile the specificity of subtiligase for the P1’ and P2’ sites (Figure 27).⁴⁶⁹ Peptide libraries derived from proteolytically digested E. coli proteome, containing nearly each of the 400 P1’-P2’ dipeptide combinations, were subjected to subtiligase-catalyzed ligation to a probe peptide and analyzed for labeling efficiency. The results indicated that the best substrates had a small amino acid, Met or Arg at the P1’ position and a large hydrophobic or aromatic residue at the P2’ position. Saturation mutagenesis of hot spots that contribute to this specificity led to the identification of subtiligase mutants with a greater substrate tolerance at these key positions. The broadened substrate scope of this library of subtiligase mutants extends the utility of the technology for the tagging and enrichment of neo N-termini in native proteomes,⁴⁷⁰ as well as for the traceless assembly of semisynthetic proteins. With respect to the latter, the Y217K mutant identified in this screen has been used for cysteine-free semisynthesis of PTEN,⁴⁷¹ (See section 6.3 Enzyme-Catalyzed Expressed Protein Ligation). As a further illustration of the tolerance of the subtilisin family to protein engineering efforts, Janssen and coworkers introduced S212C and P216A mutations into a hyperstable, calcium independent variant of subtilisin BPN’, affording a peptide ligase termed, “peptiligase”.⁴⁷² Further refinements of this system generated a variant, “omniligase”, with broad tolerance for both the P1’ and P2’ positions.⁴⁷³ In light of the recent use of subtiligase for the catalysis of cysteine-free EPL (see Section 6.3 Enzyme-Catalyzed Expressed Protein Ligation), improved subtilisin-derived peptide ligases will continue to find use as catalysts for traceless protein semisynthesis.

Figure 27. — Comprehensive characterization of subtiligase specificity by mass spectrometry.⁴⁶⁹

5.2. Trypsiligase

Trypsin has long been known to catalyze amide-bond formation under certain conditions.⁴⁷⁴ Through engineering of the structure of trypsin, Stubbs, Bordusa and coworkers have created a peptide ligase named ‘trypsiligase’ which is able to carry out both transpeptidation and ligation of peptides and peptide esters (Figure 28).⁴⁷⁵ This version of trypsin possesses four mutations that convert the protease into a transpeptidase enzyme recognizing the tri-peptide motif (-YRH-). In this case, the enzyme cleaves this motif C-terminally to the Tyr residue to form an acyl-enzyme intermediate, that may be intercepted by peptides bearing Arg-His N-terminal dipeptide sequences. This method has been used for the C-terminal labeling of the Fab region of the anti-Her2 antibody with a synthetic peptide,⁴⁷⁶ although the yield of the reaction suffered from the reversibility and hydrolysis observed for all transpeptidation reactions. Alternatively, trypsiligase accepts substrate mimetics as acyl donors, such as 4-guanidinophenyl (OGp) esters (Figure 28b),⁴⁷⁷ which can be ligated to acyl acceptors.⁴⁷⁵ Using this method, proteins including cyclophilin 18, human epidermal growth factor and parvulin 10 were modified N-terminally with various synthetic cargoes, including biotin, PEG and fluorescent dyes.⁴⁷⁵ While not yet applied beyond labeling, it will be interesting to see if this method is extended to the semisynthesis of proteins, especially those modified within their N-terminal regions, generating only a small -RH- scar. In instances where this motif is contained within the native sequence (e.g. histone H4), trypsiligase could provide one-step traceless protein semisynthesis.

Figure 28. — Trypsiligase catalyzed modification of proteins. a) C-terminal transpeptidation using trypsiligase. B) N-terminal modification of proteins using 4-guanidinophenyl esters.⁴⁷⁸

6. HYBRID CHEMOENZYMATIC LIGATION TECHNOLOGIES

The technologies described in the preceding sections are usually considered to be separate tools for protein engineering, despite sharing underlying chemistry to promote amide-bond formation, namely, enthalpic activation of the acyl donor, often as a high energy thioester intermediate, and the use of a template (i.e. the substrate binding pocket of an enzyme) to direct amide coupling. Since, as discussed, each of these activation modes comes with associated benefits and weaknesses, researchers have investigated how mixing and matching aspects of these technologies, to create what we will refer to as hybrid ligation methods, might lead to improved routes to semisynthetic proteins. Here we will briefly describe examples of these strategies.

6.1. Transpeptidase-mediated synthesis of protein thioesters

The predominant strategy for the preparation of protein α-thioesters used in semisynthesis schemes is the thiol-mediated cleavage of protein-intein fusions. As an alternative route to these reactive building blocks, the groups of Pentelute and Tam each capitalized on sequence specific transamidation reactions, affording protein α-thioesters that were used in downstream NCL reactions.^{479, 480} Pentelute and coworkers used sortase-mediated transpeptidation to install an α-thioester group into recombinant proteins bearing C-terminal sorting sequences (Figure 29a).⁴⁷⁹ Of note, the authors used this approach to insert a synthetic d-peptide sequence between two recombinant protein domains, namely the lethal factor N-terminal domain (LF_N) from anthrax toxin and the diptheria toxin α-chain (DTA). The resulting construct that was able to translocate into CHO-K1 cells and inhibit protein synthesis. Tam and coworkers used butelase 1 to create α-thioesters through high yielding transpeptidation between proteins bearing C-terminal NHV tags and glycyl-thioesters.⁴⁸⁰ This strategy was integrated into a technically impressive sequential modification process (also featuring NCL and sortase-mediated ligation steps) that allowed dual labeling of the N- and C-termini of a recombinant ubiquitin protein (Figure 29b).

Figure 29. — Transpeptidase-mediated assembly of protein thioesters in multistep ligations. a) Sortase-mediated installation of an α-thioester into a recombinant protein for use in NCL.⁴⁷⁹ LF_N, lethal factor N-terminal domain from anthrax toxin; DTA the diptheria toxin α-chain. b) Dual labelling of ubiquitin through EPL of a butelase-assembled protein a-thioester with a synthetic biotinylated peptide, followed by N-terminal sortase-mediated ligation with a fluorescein labeled peptide.⁴⁸⁰

6.2. Streamlined Expressed Protein Ligation

Split inteins have been used as a way to access protein α-thioesters in a manner that is more controlled than using their contiguous counterparts. This method, named ‘streamlined expressed protein ligation’ (SEPL), capitalizes on the ability to trigger the acyl-shift reactions in the protein splicing cascade through user-controlled reconstitution of the split intein domain.⁴⁸¹ In doing so, the premature cleavage of protein-intein fusions that can occur in vivo and during purification can be altogether avoided. Fusion proteins bearing the Int^N fragment of the Npu DnaE split intein were converted to α-thioesters through incubation with the thiol reagent MESNa and the cognate Int^C fragment, mutated at both the C-terminal asparagine (N137A) and the C-terminal extein (C+1A) residues (Figure 30).⁴⁸¹ The strong and specific interaction between the intein fragments allowed this to be performed using an immobilized Int^C fragment as a purification step. Importantly, protein α-thioesters bearing each of the 20 amino acids at the C-terminus could be isolated, with the exception of asparagine. This method has been used in the semisynthesis of histones bearing a variety of PTMs,^{155, 156, 481} the generation of antibody drug conjugates,^481–483 and to install biophysical probes into proteins.^{166, 179} Notably, the SEPL method is likely to be a direct beneficiary of the ongoing efforts to discover and engineer split inteins with improved physicochemical and biochemical properties (see section 3.3 Engineering of split inteins).

Figure 30. — Streamlined EPL using split inteins.⁴⁸¹

6.3. Enzyme-Catalyzed Expressed Protein Ligation

The successful use of NCL/EPL for protein semisynthesis is very much dependent upon the process being near traceless. However, the reaction invariably requires a cysteine or related acyl acceptor to facilitate amide-bond formation through proximity-driven entropic activation. To remove this limitation, Cole, Wells and coworkers have used the templating ability of subtiligase to enable ligation reactions between synthetic non-cysteinyl peptides and recombinant protein thioesters generated from intein-fusions (Figure 31).⁴⁷¹ Critically, the authors used this approach to generate semisynthetic PTEN in a traceless manner through the ligase-catalyzed condensation between a recombinant fragment containing a C-terminal tyrosine α-thioester and a peptide bearing an N-terminal RY dipeptide sequence.^{227, 471} Semisynthesis of PTEN using EPL previously required the installation of a non-native Cys residue for the ligation reaction, a mutation which has been found to affect the behavior of the protein in cells.^{471, 484} Using the enzyme-templated approach, Cole, and coworkers generated both monophosphorylated²²⁷ and tetraphosphorylated⁴⁷¹ forms of PTEN, which is autoinhibited by these PTMs (see section 7.5.3 for further details). It is anticipated that advances in the engineering of subtiligase and related variants (described earlier) will directly benefit this approach to protein semisynthesis, increasing both the reaction efficiency and sequence tolerance.

Figure 31. — Enzyme-catalyzed expressed protein ligation. Scheme showing intein-mediated generation of a recombinant protein α-thioester, followed by EPL with a cysteinyl peptide (upper) or subtiligase-catalyzed EPL with a non-cysteinyl peptide (lower).⁴⁷¹

6.4. Transpeptidase-Assisted Intein Ligation

Protein trans-splicing using ultra-fast split inteins offers an efficient way to re-assemble proteins from recombinant and synthetic fragments at low concentrations. However, the length of the split intein fragments, one of which must be accessed synthetically, can render this approach impractical for protein semisynthesis. Recently, it was demonstrated that the individual split intein fragments could be assembled through a prior transpeptidase mediated ligation reaction.⁴⁸⁵ Specifically, variants of the enhanced Cfa split intein, mutated to contain recognition sequences for the evolved eSrt2A sortase,⁴³² are assembled from synthetic and recombinant fragments through initial enzyme-mediated transamidation, with the resulting product then undergoing efficient PTS in situ with a complementary intein fragment fused to a protein of interest (Figure 32). A key advantage of this tandem transamidation process is that it reduces the size of the appended intein sequence to a short 7 amino acid peptide tag, which is easily accessible using standard SPPS. This strategy was used for C- and N-terminal labeling of proteins, such as Cas9,⁴⁸⁶ as well as the traceless semisynthesis of phosphorylated forms of the chromatin architectural proteins linker histone H1 and MeCP2. Further, the successful use of the methodology for the labeling of histones in chromatin in nucleo shows that it is compatible with complex cellular environments. As with other hybrid approaches, this semisynthesis technology will profit from ongoing efforts to engineer improved split inteins and transpeptidases.

Figure 32. — Generation of a C-terminally modified semisynthetic protein using transpeptidase-assisted intein ligation (TAIL).⁴⁸⁵

7. APPLICATIONS OF PROTEIN SEMISYNTHESIS

The development of new and improved strategies for protein semisynthesis has been driven by the need to understand protein structure and function. Many areas of biological inquiry have benefited greatly from the precision generation of modified proteins, using both chemical and enzymatic approaches. In this section we highlight some key areas where protein semisynthesis methods have helped fuel biological discovery, with emphasis placed on developments that have occurred in the last decade.

7.1. Chromatin modifications

Eukaryotic genomes are packaged into a dense nucleoprotein complex known as chromatin. Like beads on a string, the DNA is spooled into small repeating units known as nucleosomes.⁴⁸⁷ Each nucleosome contains around 147 bp DNA wound around an octameric complex of histone proteins (two copies each of H2A, H2B, H3 and H4). The disordered N- and C-terminal tails of histones that protrude from the nucleosome core have long been known to serve as hubs for a plethora of PTMs (Figure 33), which have been correlated with the transcriptional state of the underlying genomic locus.⁴⁸⁸ Accordingly, unraveling the complex biochemical and biophysical implications of histone modifications (colloquially referred to as ‘marks’) has become a dynamic and cross-disciplinary area of inquiry, in which peptide and protein synthesis have played pivotal roles (for detailed accounts that emphasize the important role of chemistry in the area, we direct the reader to the following reviews^489–494).

Figure 33. — Histone modifications. a) Structure of a mononucleosome (PDB ID: 1KX5). b) Primary structure of histone tails with important modifications annotated. Ac, acetyl; Cit, citrullyl; Me, methyl; Ph, phosphoryl; Ub, ubiquityl.⁴⁹¹

7.1.1. Histones

The generation of modified histone proteins by protein semisynthesis has been especially fruitful for many reasons, the proximity of most PTMs to the N- and C-termini, the ability of histones to be refolded (allowing the use of chaotropes in assembly reactions), and the general lack of endogenous cysteine residues. This last feature has allowed cysteine-specific chemistry to be used in conjunction with amide-bond formation (such as radical desulfurization,³³⁷ sulfhydryl-alkylation^{165, 495} or disulfide formation^{496, 497}) to create completely native sequences or close mimics thereof. In a pioneering study that employed an experimental workflow broadly adopted in the many studies that followed (Figure 34a), Peterson and coworkers used semisynthesis to generate ‘designer’ chromatin containing a specific histone PTM, in this case histone H3 phosphorylated at position S10 (H3S10p), and then used this reagent to gauge how the mark impacted downstream chromatin biochemistry.⁴⁹⁸ The desired semisynthetic protein was assembled from a synthetic peptide α-thioester spanning residues 1-31 of histone H3 and a recombinant truncated histone spanning residues 32-135, bearing a Thr32Cys mutation necessary for the ligation reaction. The purified semisynthetic protein was refolded into histone octamers with recombinant histones H2A, H2B and H4, followed by deposition onto a DNA fragment containing a tandem array of nucleosome positioning sequences. These nucleosome arrays were then assayed for phosphorylation-dependent effects on chromatin remodeling by the SWI-SNF complex, as well as effects on the acetyltransferase activity of the Gcn5-containing SAGA complex. In contrast to previous studies that employed histone peptide substrates,^{499, 500} the acetyltransferase activity of the SAGA complex was not stimulated by phosphorylation in a chromatin context, highlighting the importance of using these more physiologically relevant substrates. In the same year, McCafferty and coworkers used a similar NCL-based strategy to prepare polyacetylated (K4Ac, K9Ac, K14Ac, K18Ac and K23Ac) and methylated (K9me3) forms of histone H3, as well as polyacetylated (K5Ac, K8Ac and K12Ac) histone H4.¹⁶¹ Importantly, the authors were able to achieve completely traceless semisynthesis of these proteins through a post-ligation Cys-to-Ala conversion at the ligation junction by means of hydrogenolytic desulfurization (Figure 34b).³³⁶ In the years following these first studies, the combination of chemical ligation and cysteine desulfurization (typically now by radical means)³³⁷ has been extensively used for the traceless semisynthesis of N-terminally (and C-terminally) modified forms of all four histones (see Table 1). Indeed, histones are without question the most heavily studied proteins using the semisynthesis paradigm.

Figure 34. — Semisynthesis applied to histone H3. a) Semisynthesis of H3Ser10ph through NCL, and incorporation into nucleosome arrays (Shogren-Knaak *et al.*).⁴⁹⁸ b) Traceless semisynthesis of polyacetylated H3 using NCL and desulfurization (He *et al.*).¹⁶¹ c) Traceless semisynthesis of H3 by sortase-mediated ligation using the F40 sortase.

Sortase-mediated ligation has also been used to access semisynthetic histone H3.^{433, 436, 437} The engineered “F40” sortase recognizes residues 29-33 of histone H3 (APATG) thereby allowing assembly of the native protein from a synthetic peptide, spanning residues 1-33 of histone H3 (containing the non-canonical sortase sequence), and a truncated recombinantly expressed form of histone H3 (33-135) (Figure 34c). Cole and workers used this strategy to prepare nucleosomes containing semisynthetic H3K9Ac as substrates for the CoREST transcriptional repressor in an effort to develop dual action inhibitors against the HDAC and demethylase activities within this multi-protein complex.⁴³⁷ In order to increase the yield of the reaction, this same group subsequently used synthetic glycolate-containing depsipeptides as a way of overcoming the reversible nature of the transpeptidation reaction.⁴³⁵ The increased efficiency associated with this adjustment allowed for the production of various semisynthetic H3 proteins, combinatorially modified with dimethylation at K4 and acetylation at K9, K14 and/or K18. These modified proteins were subsequently used to interrogate the rates of demethylation and deacetylation by the CoREST complex. Although sortase-mediated semisynthesis of H3 cannot currently provide direct access to modifications beyond residue 33, this method has many benefits over other strategies including NCL and desulfurization, by providing access to the native protein sequence in a single step.

Semisynthetic histones bearing C-terminal modifications have been accessed using a combination of EPL and desulfurization chemistry.^{155, 157, 159, 174, 501} In the first synthesis of ubiquitinated H2B, a synthetic peptide spanning residues 118-125 of the protein was ligated through the side-chain of K120 using an auxiliary-mediated ligation, to a recombinant ubiquitin α-thioester produced from thiolysis of an intein fusion (Figure 35a).³⁴⁵ Photolytic removal of the auxiliary and a protecting group masking the N-terminal cysteine afforded an ubiquitinated peptide that was then ligated to a recombinant protein α-thioester spanning residues 1-116 of the histone, accessed through intein thiolysis. Desulfurization of the non-native Cys residue afforded the native ubiquitinated protein. Subsequent biochemical assays with mononucleosomes containing H2BK120ub demonstrated that this modification stimulates the methyltransferase activity of the catalytic domains of Dot1¹⁵⁹ and Set1^502–504 (Figure 35b). A revised semisynthesis of ubiquitinated H2B replaced the use of a thiol auxiliary with a Cys residue, effectively creating a G76C mutation of ubiquitin upon ligation that, upon desulfurization, afforded a G76A scar.⁵⁰⁵ More facile access to ubiquitylated histone H2B, either through this method or through disulfide mimetics, has allowed detailed studies into the role of the PTM in methyltransferase stimulation,^{310, 496, 506} as well as its impact on chromatin stability and structure.^{157, 497, 507} This streamlined synthetic approach was also adopted for the semisynthesis of ubiquitinated H2A (H2AK119ub),¹⁵⁷ following a similar strategy of dual EPL reactions and subsequent desulfurization, although penicillamine was used as a surrogate for Cys in the second ligation reaction, which was converted to a native Val through desulfurization. Subsequently, auxiliary-directed ligation approaches have been revisited to achieve traceless semisyntheses of H2AK119ub,¹⁵⁸ as well as the DNA damage associated H2AK13ub and H2AK15ub.⁸¹ It should be stressed that this histone ubiquitination work has stimulated a tremendous amount of peptide and protein chemistry research, studies that are further motivated by the multifarious roles of ubiquitin in eukaryotic biology and the need to access ubiquitinated polypeptides for biochemical studies. Indeed, there have been several highly impactful methodological advances in this area, which have collectively advanced the ubiquitin field greatly. The interested reader is referred to several excellent reviews on this topic.^508–511

Figure 35. — Semisynthesis applied to ubiquitinated histone H2B. a) Scheme for the semisynthesis of ubiquininated histone H2B from three fragments.¹⁵⁹ b) PTM crosstalks biochemically verified using semisynthetic ubiquitinated histone H2B.

An additional improvement to the generation of modified histones came courtesy of the streamlined EPL protocol,⁴⁸¹ which as discussed earlier uses ultrafast split inteins for the generation of protein α-thioesters. This method was used in the preparation of an α-thioester of H2B (residues 1-116) subsequently employed in the semisynthesis of H2BK120Ac. Fierz and coworkers adopted the SEPL approach for the impressive semisynthesis of the histone variant H2A.X phosphorylated at serine 139 (known as γH2A.X) and ubiquitinated at K15 (Figure 36),¹⁵⁶ a combination of histone PTMs that is important for the DNA-damage response.⁵¹² The remote positions of these modifications, on both the N- and C-termini, required that the authors assemble the histone sequence from three pieces, a synthetic C-terminal cysteinyl peptide spanning residues 135-142 (containing S139ph), a recombinant central portion spanning residues 21-134, and a K15 ubiquitinated N-terminal fragment containing residues 1-20 (generated itself through EPL between a recombinant ubiquitin α-thioester and a synthetic peptide). In order to mask reactive functionalities, the authors expressed the central recombinant portion as an N-terminal SUMO fusion and a C-terminal fusion with the Npu^N DnaE split intein. The ligation of this construct to the C-terminal fragment was first achieved through co-incubation in the presence of the complementary Npu^C fragment, and additional thiols MESNa and methylthioglycolate, which formed the reactive α-thioester in situ. Following the ligation, treatment with Ulp1 protease unmasked the γH2A.X (21-142) bearing an N-terminal cysteine for the second ligation reaction with the ubiquitinated N-terminal semisynthetic peptide hydrazide. Following ligation and desulfurization, the purified proteins were incorporated into chromatin carrying FRET pairs, and the conformation analyzed on the single-fiber level through total internal reflection fluorescence (TIRF) imaging. Accordingly, it was found that while S139ph does not directly alter chromatin structure, the presence of K15 ubiquitination impairs the formation of higher order chromatin folding.

Figure 36. — Semisynthesis of ubiquitinated and phosphorylated histone H2A.X.¹⁵⁶

The discovery of many non-canonical histone modifications has driven the development of new chemistry to access modified nucleosomes. While long known to be ubiquitinated, histone proteins are also modified through SUMOylation,⁵¹³ with H4K12 sumoylation (H4K12su) being associated with gene repression.⁵¹⁴ Chatterjee and coworkers generated H4K12su semisynthetically through a sequence of auxiliary-mediated EPL reactions between a synthetic peptide α-hydrazide and a recombinant SUMO-3 α-thioester (generated from a Mxe GyrA intein fusion), the product of which was then ligated to recombinantly expressed truncated H4.¹⁷⁶ Reduction of both the 2-(aminooxy)ethanethiol ligation auxiliary³⁴⁴ and the reactive cysteine (through desulfurization) at various points in the synthesis generated the native sumoylated protein. Interestingly, nucleosomes containing both H4K12su and H3K4me2 were preferred substrates for demethylation by the CoREST complex, dependent on a functional SUMO-interacting motif in CoREST that is proposed to recruit the complex proximal to the H3 tail.¹⁷⁶ The synthetic strategy used for the synthesis of H4K12su has been similarly applied to the semisynthesis of sumoylated H2B, although the functional consequences of this modification are yet to be uncovered.¹⁶⁰

The covalent attachment of the neurotransmitter serotonin was recently found to occur on Gln5 of histone H3,¹⁶⁵ catalyzed by the enzyme tissue transglutaminase 2 (TGM2). In order to test whether Q5 serotonylation can affect the deposition of methyl marks at the proximal H3K4 site, semisynthetic serotonylated H3 was generated through NCL (Figure 37). A synthetic peptide α-thioester spanning residues 1-13 of H3 was synthesized through the thioester-generating SEA-linker,⁵⁹ and serotonin coupled to an orthogonally protected glutamate residue at position 5 to generate the serotonylated glutamine. Cleavage from the resin afforded a crude serotonylated SEA-functionalized peptide that was converted to a mercaptopropionyl thioester through N-to-S acyl shift and transthioesterification. This peptide was subjected to NCL with a recombinantly produced H3(14-135) bearing an N-terminal cysteine, followed by cysteine alkylation with bromoethylamine³²⁹ to give the ligated product with a pseudolysine mimic in place of the native lysine at position 14. Unexpectedly, the presence of the bulky serotonin moiety at H3Q5 did not affect methylation of H3K4 in nucleosomes by the MLL1 methyltransferase complex. Likewise, nucleosomes either unmethylated or trimethylated at H3K4 were equally modified with serotonin by TGM2, shedding light on the observed coexistence of these marks in cells.¹⁶⁵

Figure 37. — Semisynthesis of serotonylated histone H3 using NCL followed by cysteine alkylation.¹⁶⁵

7.1.2. Asymmetrically Modified Nucleosomes

Nucleosomes have intrinsic two-fold pseudo-symmetry, reflecting the fact that there are two copies of each histone in the nucleoprotein complex.⁴⁸⁷ In vitro reconstitution of nucleosomes from recombinant and/or semisynthetic histones typically results in retention of this symmetry, since the two copies of each histone in a nucleosome will contain the same modification(s). This is a property that is not altogether shared with nucleosomes in vivo, for example, so-called bivalent domains at developmentally regulated promotors contain nucleosomes bearing both activating (H3K4me3) and repressive (H3K27me3) marks on separate tails.⁵¹⁵ Accordingly, efforts have recently been directed to the generation of asymmetrically modified nucleosomes to inform on biochemical cross-talk,^515–518 or asymmetric processing mechanisms by remodeling enzymes.^{519, 520} Initial efforts to generate asymmetric nucleosomes involved refolding of mixtures of modified histones into octamers, followed by two-step affinity purification through non-native tags to generate asymmetrically modified octamers that could be reconstituted onto DNA templates.⁵¹⁵ More recently, Fierz and coworkers designed a method to produce nucleosomes containing asymmetrically modified histone H3 through the use of traceless chemically tethered precursors (Figure 38a).^{516, 518} Semisynthetic H3 proteins, trimethylated at K4, K27 or K36 were fused as asymmetric disulfides through short cysteine containing N-terminal linker sequences. Nucleosomes formed from refolded histone octamers containing these tethered constructs could then be converted in a traceless manner into the native asymmetric nucleosomes through reduction of the disulfide tether and liberation of the linkers with TEV protease. Importantly, these native bivalent nucleosomes validated previous studies suggesting that stimulation of the H3K27 methyltransferase activity of the enzyme PRC2 could be achieved intranucleosomally,⁵¹⁵ and that interference of H3K27 methylation by pre-existing H3K4 or K36 methyl marks can only occur on the same histone tail. Notably, these reagents have also proven extremely useful for the development of multivalent probes that specifically sense bivalent nucleosomes in cells.¹⁶² This tethering strategy for generating asymmetrically modified nucleosomes has been extended to histone H4, in this case using tethering sequences linked to a lysine side-chain in order to access the natively N-terminally acetylated protein.⁵¹⁷ In this way, the authors were able to observe the distributive mechanism of H4K20 methylation by the methyltransferase Set8. Bowman and coworkers have generated asymmetrically modified nucleosomes through the stepwise association of H2A/H2B dimers onto the asymmetric Widom 601 DNA sequence, which is commonly used in nucleosome constitutions (Figure 38b).⁵²¹ Observing preferential association between H2A/H2B dimers and the TA-rich half of the 601-sequence, the authors were able to purify hexasomal species containing one H2A/H2B dimer, to which another dimer could then be added to complete the nucleosomal structure.⁵¹⁹ By generating highly oriented nucleosomes containing a single ubiquitinated H2B, the authors were able to show that this modification can stimulate the activity of the chromatin remodeler Chd1, but only when present on the entry side of the nucleosome (i.e. the side positioned to bind DNA that is pulled onto the nucleosome through remodeling activity). This strategy has also proven useful for the asymmetric labeling of nucleosomes with fluorescent tags, allowing single-molecule FRET analysis of the nucleosome remodeling process.⁵²² We expect that both of these complementary approaches will find further utility in the dissection of the fine molecular mechanisms underpinning the asymmetric sensing of modified nucleosomes and nucleosome arrays by chromatin factors.

Figure 38. — Strategies for the synthesis of asymmetrically modified nucleosomes. a) Reconstitution of asymmetric nucleosomes containing bivalent H3K4me3 and H3K27me3 modifications using traceless disulfide-tethering of semisynthetic H3 proteins.^{516, 518} b) Stepwise incorporation of histone tetramers and dimers onto the Widom 601 nucleosome positioning DNA sequence.⁵²¹

Methodological advances have greatly simplified the preparation of modified histone proteins, however elucidating the biochemical effects of each modification remains a daunting task. Moreover, the compounding effects of post-translational modifications, histone variants, disease-associated mutations,⁵²³ not to mention DNA modifications,^{524, 525} creates an overwhelming number of nucleosomal substrates to test individually. The use of DNA-barcoding has allowed for such biochemical experiments to be performed simultaneously on a library of uniquely modified nucleosomes, each identifiable using DNA-sequencing (Figure 39).⁷⁷ This nucleosome library was first used to profile binding specificity of histone reader proteins, stimulation of p300 acetyltransferase activity in vitro, and multiple PTM cross-talk relationships on a systems level through incubation with an active nuclear extract. Understanding the substrate preference of the ISWI family of chromatin remodeling enzymes was achieved using an expanded 115-member library of modified mononucleosomes.¹⁵⁵ Using a previously established assay for chromatin remodeling that is coupled to DNA cleavage by a restriction enzyme, the kinetics of remodeling were measured for 6 unique complexes, which were shown to display differing preferences for the modification state of a nucleosome. Critically, the nucleosome acidic patch, a binding site used by many chromatin binding proteins enzymes,⁴⁸⁷ was found to be an important regulatory epitope for each of the remodelers. Substrate-profiling using this library has been subsequently used for histone ADP-ribosylation by poly(ADP-ribose) polymerase (PARP) enzymes⁵²⁶ and ubiquitination of H2B by the UBE2A:RNF20/40 ligase machinery.⁵⁰¹ In both cases, novel modes of enzyme regulation were revealed, specifically an inhibitory role for H3K9 acetylation in the case of PARP-mediated H3S10 ADP-ribosylation and a stimulatory role of for H2A tail in the case of H2B ubiquitination.

Figure 39. — DNA-barcoded nucleosome library for high-throughput chromatin biochemistry.⁷⁷ Next-generation DNA sequencing allows for experiment multiplexing and quantitative read-out of individual nucleosome library members.

7.1.3. Chromatin-Associated Proteins

Protein semisynthesis has been used in the study of chromatin-associated proteins other than histones (Table 1). As an example, Fierz and co workers used SEPL to assemble obligate dimers of HP1α,¹⁷⁹ a chromatin effector that binds H3K9me3 and induces chromatin compaction.⁵²⁷ This was achieved using an affinity-directed dual EPL approach that resulted in the C-terminal chromo-shadow domain in each copy of the protein being linked via a peptide adaptor derived from the protein shugoshin,⁵²⁸ which has high affinity for the HP1α dimer. The authors found that this covalent dimer had both prolonged binding to chromatin containing semisynthetic H3K9 methylation, as well as increased binding frequency as measured by single molecule TIRF microscopy, indicating that multivalency of HP1α is an important feature for fast and efficient binding to H3K9me3 containing heterochromatin domains.¹⁷⁹ In other work, Margueron and coworkers used NCL to generate a methylated form of Jarid2, a cofactor of the PRC2 methyltransferase complex that is itself modified by this enzyme.¹⁸⁹ A synthetic peptide α-thioester containing residues 109-123 (containing K116me3) was ligated to a recombinant fragment spanning residues 124-450 to give a semisynthetic fragment of the Jarid2 protein. Interestingly, methylation at this position was shown to bind the EED subunit of PRC2 and stimulate the activity of the catalytic EZH2 methyltransferase subunit of the complex. A recent study, also using semisynthetic Jarid2 fragments, has shown that this stimulation of PRC2 activity on chromatin can be modulated by the presence of specific phosphoserine residues near the site of Jarid2 methylation.¹⁹⁰ As another example in the area of chromatin-associated proteins, split DnaE inteins have been used to attach various synthetic cargoes to a nuclease dead version of CRISPR-Cas9 (dCas9) which were subsequently delivered into living cells in complex with guide RNAs for genomic-targeting.⁴⁸⁶ This system was used to recruit various epigenetic factors to user-defined loci, taking advantage of the availability of small molecule binders to component domains.

7.1.4. Semisynthetic Modification of Native Chromatin

With the help of ultra-fast split inteins, the semisynthesis of histones has been extended to a native chromatin setting.^{397, 401, 506} In a semisynthesis of H2B ubiquitinated on K120, histone H2B (1-116) was expressed in HEK 293T cells as a fusion to the Int^N fragment of the Ava DnaE split intein (Figure 40).⁴⁰¹ Isolated nuclei from these cells were then treated with a semisynthetic ubiquitinated H2B peptide (117-125) fused to a complementary Int^C fragment, generating the semisynthetic protein through PTS on chromatin. The presence of this mark was sufficient to induce the upregulation of H3K79 methylation, a crosstalk that has been well-characterized in vitro.¹⁵⁹ Notably, the ability to manufacture ubiquitinated H2B in nucleo proved instrumental in a subsequent mechanistic dissection of this crosstalk that led to the identification of a functional epitope on the ubiquitin surface required for stimulation of the H3K79 methyltransferase Dot1.⁵⁰⁶ Access to N-terminally modified histones by PTS in nucleo has also been achieved through the use of the promiscuous Cfa_GEP DnaE split intein.³⁹⁷ Histone H3 containing the K27me3 heterochromatin mark was generated through the treatment of cell nuclei expressing histone H3 with the Int^C fragment embedded in the N-terminal tail with a semisynthetic construct corresponding to the Int^N fragment fused to H3 (1-28) containing K27me3.

Figure 40. — Semisynthesis of ubiquitinated histone H2B in isolated nuclei.⁴⁰¹

The ability to modify chromatin in nucleo can be considered an intermediate step between in vitro reconstitution of designer chromatin, which is now a fairly established field,⁴⁸⁹ and the ultimate goal of chemically customizing chromatin in a locus specific manner in living systems.⁵²⁹ While there has been some progress in performed histone semisynthesis in living cells, by delivering split intein fusions into transfected cell lines,⁴⁰¹ this does not provide control over genomic location. Efforts to achieve this, perhaps involving the genomic targeting capabilities of dCas9, will certainly test the ingenuity of chemical biologists in the future. However, the great promise such strategies hold for validating causal relationships attendant to epigenetic control of gene expression (and other genomic transactions) argues for this being a worthy undertaking.

7.2. Toxic Proteins in Neurodegenerative Disease

A broad range of neurodegenerative disorders, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis and prion diseases, share a common feature of accumulation of toxic protein aggregates. The molecular mechanisms behind the aggregation of specific proteins that are linked to these diseases are not altogether understood. Of particular interest are the roles of the various PTMs that decorate these proteins, a question that is, in principle, well suited to the protein semisynthesis approach. In this section, we discuss how semisynthesis has been deployed in this area, allowing the generation of modified forms of these proteins in order to understand the molecular mechanisms behind the neurotoxicity.

7.2.1. α-Synuclein

α-Synuclein is a 140 amino acid protein that is abundantly expressed in the nervous system.⁵³⁰ The aggregation of α-synuclein into intracellular inclusions is a hallmark of Parkinson’s disease (PD) and related neurodegenerative “synucleinopathies”.^{531, 532} A number of PTMs have been discovered on α-synuclein aggregates isolated from PD patients, including phosphorylation,⁵³³ glycosylation,⁵³⁴ ubiquitination⁵³⁵ and nitration.⁵³⁶ The small, unfolded nature of α-synuclein makes total synthesis and semisynthesis useful for the study of this protein (further reviewed elsewhere⁵³⁷). In particular, semisynthetic approaches centered on the use of NCL and EPL have been used to access phosphorylated,^{284, 286–288} ubiquitinated,^282,283 glycosylated,^{289–291, 538} nitrated²⁸⁵ and N^α-acetylated²⁸¹ forms of this protein, as well as α-synuclein containing a backbone thioamide which served as a fluorescent quenching probe to study conformational changes during aggregation.²⁸⁰

To prepare C-terminally modified α-synuclein, Lashuel and coworkers used EPL coupled with cysteine desulfurization to install a phospho-tyrosine at position 125 (Y125ph) of the protein (Figure 41a).²⁸⁴ A protein α-thioester spanning residues 1-106 of α-synuclein, generated from thiolysis of a recombinant fusion with the Mxe GyrA intein, was ligated to a synthetic peptide spanning residues 107-140 bearing the phosphotyrosine. Conversion of the reactive Cys107 residue to the native Ala107 was subsequently achieved by radical desulfurization, affording the Y125ph modified protein, which displayed no significant difference in aggregation properties relative to the unmodified protein. By contrast, semisynthetic α-synuclein containing phosphoserine at position 129, generated through the same strategy, was more prone to aggregation and exhibited decreased membrane binding, supporting a role for this modification in the pathogenesis of PD.^{286, 287}

Figure 41. — Semisyntheses of modified forms α-synuclein, including a) C-terminal,²⁸⁴ b) N-terminal,²⁸² and c) central modifications.²⁸⁹

The role of ubiquitination, and the related SUMOylation, in regulating α-synuclein aggregation has been heavily studied using both protein bioconjugation and semisynthesis approaches. In an example of the latter, Lashuel, Brik and coworkers used a combination of NCL and EPL to assemble an N-terminally ubiquitinated version of α-synuclein from three-fragments (Figure 41b).²⁸² In this case, truncated α-synuclein (residues 19-140) containing an N-terminal Cys, accessed directly from recombinant expression in E coli, was ligated to a synthetic peptide α-thioester generated through Boc-strategy SPPS. A δ-mercaptolysine building block was incorporated at position 6 to direct ubiquitination through EPL with a recombinantly expressed ubiquitin α-thioester. Radical desulfurization then afforded the native K6 ubiquitinated α-synuclein. Interestingly, the presence of ubiquitin at this position inhibited α-synuclein aggregation in vitro without altering the membrane-binding affinity, suggesting a protective role for this modification. Impressively, this synthetic strategy has been applied to the semisynthesis of both di- and tetraubiquitinated α-synuclein at K12,²⁸³ work that highlights the extraordinary capabilities of modern protein ligation technologies. Other studies in this area, involving the use of streamlined cysteine-based ubiquitination/SUMOylation conjugation strategies,^{496, 539} have allowed systematic analyses of these modifications as a function of attachment site on α-synuclein.^540–542 This has revealed that the effect of ubiquitination on the aggregation and proteosomal stability of the protein is strongly position-dependent, underscoring the nuanced roles of PTMs in regulating protein function.^540–542

In another impressive body of work, Pratt and coworkers used semisynthesis to generate a series of α-synuclein variants containing the O-linked monosaccharide, N-acetyl glucosamine (GlcNAc).^{289–291, 538} These modifications are located within the center of the α-synuclein and require a combination of NCL and EPL reactions to assemble the protein sequence from three fragments (Figure 41c). The strategy taken is illustrated through the assembly of α-synuclein containing GlcNAc at serine 87, in which the protein was assembled from a synthetic peptide α-thioester containing a preinstalled Ser-GlcNAc residue, and two recombinant fragments spanning residues 1-75 and 92-140 respectively. Following sequential ligation reactions, desulfurization of the reactive cysteines afforded the native protein. Using this approach, it was found that attachment of the sugar at S87 and T72 inhibits protein aggregation, without affecting its endogenous membrane binding affinity. Access to a panel of glycosylated forms of α-synuclein has allowed this group to delve deeply into the roles of this modification in regulating the biochemical and biophysical behavior of the protein.

7.2.2. Tau

Tau is a microtubule-associated protein responsible for the stabilization of axonal filaments in neurons.⁵⁴³ Hyperphosphorylation of tau leads to dissociation from microtubules and the formation of insoluble aggregates that are key features of Alzheimer’s disease and other tauopathies.⁵⁴⁴ Schwarzer, Hackenberger and coworkers used EPL to access tau bearing a C-terminal phosphorylated serine residue (S404ph) (Figure 42a).²⁹⁵ The authors ligated a synthetic phosphopeptide spanning the C-terminal 52 amino acids of tau to a recombinant α-thioester fragment comprising residues 1-389 of the protein, generated through thiolysis of a Mxe GyrA intein fusion. In this case, an Ala390Cys mutation was required for the ligation reaction, which did not affect the function of the semisynthetic proteins based on the ability to promote polymerization of tubulin in vitro. Semisynthetic tau bearing S422ph was accessed in a similar fashion, the aggregation properties of which could be neutralized by an antibody against this known pathological PTM.²⁹⁶ Difficulties in the purification of the semisynthetic protein prompted Hackenberger and coworkers to use a photocleavable biotinylated linker as a traceless purification handle,²⁹³ which was employed in the semisynthesis of tau carrying a Ser400GlcNAc modification.²⁹⁴

Figure 42. — Semisyntheses of modified versions of the tau protein.

Lashuel and Haj-Yahya developed a multi-step semisynthetic route to tau amenable to accessing all modifications within the central microtubule-binding domain (Figure 42b).⁴⁹ The N-terminal fragment, covering residues 2-245, was accessed through recombinant expression and the standard intein thiolysis workflow. Four synthetic fragments were accessed through Fmoc-strategy SPPS, covering residues 246-441. Assembly of the five fragments was achieved through convergent EPL and NCL reactions, incorporating strategic desulfurization steps to selectively convert non-native cysteine residues to the native alanines. The authors used this strategy to synthesize tau proteins bearing an acetyl-lysine at K280, a phosphotyrosine at Y310, and dual phosphoserines at S396 and S404. Tau bearing K280Ac was shown to have enhanced aggregation kinetics relative to the unmodified protein. Most recently, Becker and coworkers developed a semisynthetic route to tau that also allowed access to microtubule binding region of the protein.²⁹⁷ In this case, a three-piece strategy was employed featuring two flanking recombinantly derived fragments and a central synthetic peptide insert (Figure 42c). This route allowed access to variants of the protein containing phosphoserines and an unusual carboxymethyllysine modification. The latter was found to inhibit tubulin polymerization without affecting aggregation, whereas phosphorylation was found to inhibit aggregation without affecting tubulin binding. Collectively, these semisynthetic strategies provide good coverage of the entire C-terminal half of the tau protein and, as a consequence, will greatly aid biochemical and biophysical investigations of this important protein.

7.2.3. Prion Protein

Semisynthesis has been used to generate modified forms of the cellular prion protein (PrP), misfolding of which causes neurological diseases.^{545, 546} In early work, Becker and coworkers explored two semisynthetic routes to PrP bearing C-terminal palmitoyl groups as mimics of the native glycosylphosphatidylinositol (GPI) anchor.²³⁶ The authors first used EPL to assemble the semisynthetic protein, ligating a recombinant PrP α-thioester (residues 90-232, accessed from intein thiolysis) to synthetic palmitoylated peptides (Figure 43a). In the same study, the authors also investigated a PTS-based approach to assemble modified PrP from a recombinant PrP-fused to the Int^N fragment of the Ssp DnaE split intein and a synthetic peptide containing the cognate Int^C fragment and a short palmitoylated peptide sequence.²³⁶ This initial work was soon followed by the semisynthesis of the native GPI-anchored protein (Figure 43b).²³⁵ This collaborative work between the Becker and Seeberger groups stands as milestone in the field since it combines cutting-edge approaches in protein semisynthesis and carbohydrate synthesis. Access to these variously lipidated versions of PrP yielded a number of insights into the role of the modification, including how it alters the interaction of the protein with membranes leading to a disruption of membrane integrity.²³⁴

Figure 43. — Semisynthesis of the prion protein (PrP). a) Strategy for semisynthesis of C-terminally palmitoylated PrP.²³⁶ b) Structure of semisynthetic PrP bearing a native GPI anchor.²³⁵ c) Semisynthesis of PrP bearing glycan mimics at positions 181 and 197.³³⁸

Li and coworkers reported a different strategy for the semisynthesis of unmodified murine PrP from three fragments.²³³ An N-terminal recombinant protein α-hydrazide was generated through hydrazinolysis of a chemically cyanylated Cys-containing precursor.⁵⁴⁷ This recombinant α-hydrazide was converted to an α-thioester in situ and ligated to a synthetic sequence spanning residues 178-230 of mPrP (itself generate from two smaller synthetic fragments). This semisynthetic strategy makes use of the two native Cys residues in the PrP sequence for ligation reactions. However, as one of these junctions comprises a -DC- dipeptide sequence (requiring the use of an unstable Asp α-thioester), Li and coworkers were obliged to mutate this Asp residue to Ser.²³³ In an alternative approach, Araman and coworkers instead disconnected the protein at an -HD- dipeptide junction,²³⁷ which could be reassembled using a combination of EPL and selective desulfurization from a recombinant protein α-thioester and a peptide bearing a β-mercapto Asp building block as a Cys surrogate (Figure 43c).³³⁸ The recombinant PrP sequence containing residues 23-177, thiolyzed from a Mxe GyrA intein fusion, was ligated to synthetic peptides spanning residues 178-231 bearing the N-terminal β-mercapto Asp as well as pegylated Asn residues 181 and 197 as mimics of large N-linked glycans at these positions. Selectively desulfurized ligation products (with the native cysteine residues intact) displayed less α-helicity when modified at both Asn181 and Asn197, and all modified PrP variants were able to inhibit the in vitro formation of wild type aggregates at sub-stoichiometric levels.

7.2.4. Huntingtin

The huntingtin protein has been heavily implicated in Huntington’s disease, an inherited disorder characterized by progressive neuronal degeneration.⁵⁴⁸ The precise physiological function of the protein is still unclear, however, individuals with the disease contain a genetic polymorphism in the huntingtin gene that results in a repeat expansion of glutamine residues in the N-terminal region of the protein.⁵⁴⁹ Lashuel and coworkers have used NCL to gain semisynthetic access to this biomedically important protein (Figure 44).¹⁸² To this end, a recombinant N-terminally truncated fragment (residues 10-90) was expressed with an Ala10 to Cys mutation and ligated to a synthetic peptide bearing a phosphorylated threonine at position 3 and a C-terminal N-acyl urea as a thioester equivalent. Following ligation, the full-length protein was desulfurized in order to access the native sequence at the ligation junction. Interestingly, the presence of phosphorylated threonine significantly slowed the oligomerization of huntingtin into fibrils. This same strategy was then adapted to the semisynthesis of a mutant huntingtin protein, containing an expanded polyQ tract (43Q).¹⁸³ Similar to the wild-type protein, Thr3 phosphorylation strongly inhibited the aggregation and fibril formation of the semisynthetic mutant huntingtin, although this inhibitory effect could be reversed by the presence of acetylation at Lys6. In addition, subsequent work from Lashuel and coworkers have shown that phosphorylation at Ser13 and Ser16 can also inhibit the aggregation of mutant huntingtin.¹⁸⁴ This suggests that crosstalk between these and possible other modifications on the huntingtin N-terminus may influence the structure and aggregation properties of this protein.

Figure 44. — Semisynthesis of phosphorylated huntingtin protein (Htt).¹⁸²

7.3. Integral membrane proteins

As many as 30% of the proteins encoded in the human genome are predicted to be embedded in cellular membranes.^550–552 The biomedical importance of these proteins can hardly be overstated, fully 60% of clinically prescribed drugs target membrane proteins such as G-protein coupled receptors (GPCRs), receptor kinases and ion channels,^{553, 554} It is equally hard to overstate the technical challenges associated with studying this class of protein – as but one measure of this, only 3.6% of structures in the protein data bank⁵⁵⁵ as of June 2019 were membrane proteins (although we note that this situation is improving due to technical advances in crystallography and electron microscopy). Extending the range of protein semisynthesis into this arena is enormously challenging due to difficulties associated with preparing and manipulating hydrophobic membrane protein fragments, not to mention the undertaking required to fold these polypeptides into a native structure embedded in a membrane. Nonetheless, there have been a number of remarkable successes in this area, that have yielded important insights into membrane protein function, and that hopefully serve as a beachhead for more work in this area.

7.3.1. Ion Channels

Ion-channels are integral membrane proteins that serve as conduits for ions to pass into and out of cells. This large family of proteins is diversified by the type of ion they conduct and by the regulatory inputs that open and close their conductive pores. Potassium channels exhibit an extremely high degree of selectivity for the K⁺ ion over other cations, dictated by a stretch of conserved amino acids that form an ion-binding region known as the “selectivity filter”.⁵⁵⁶ While high-resolution structural studies indicate a key role for the protein backbone in the function of the filter, testing these predictions using conventional mutagenesis is not straightforward. This motivated the development of a semisynthetic route to the archetypical bacterial potassium channel, KcsA, an undertaking that required solving several difficult technical problems along the way, including how to fold the semisynthetic protein into the active homo-tetramer.^{102, 191}

Critically, the optimized semisynthesis that was developed provided chemical access to the selectivity filter (Figure 45a–b), allowing backbone engineering of this region, namely replacement of a key amide with an ester moiety¹⁹³ and the substitution of a glycine with a D-Ala.^{194, 557} In both cases, high-resolution crystal structures of the semisynthetic proteins were obtained to complement the functional data (Figure 45c–d). The work on the D-Ala containing KcsA analog is especially noteworthy, since it established a key role for conformational changes in the filter in ion selectivity. In a related study, Valiyaveetil and coworkers assembled the 282-residue archaebacterial voltage-dependent K⁺ channel, KvAP, from three fragments, two recombinant and one synthetic (Figure 46).¹⁹² This involved two sequential ligation reactions, again allowing synthetic access to the selectivity filter. In this case, incorporation of a D-Ala in place of a glycine offered insights into the structural basis of so-called C-type inactivation of the channel. More recently, a semisynthetic version of KcsA was prepared containing site-specific heavy isotopes in the selectivity filter.^{197, 558} This allowed two-dimensional infrared spectroscopy to be applied to the system, providing insights into the conformational transitions that occur in the filter during multi-ion conduction.

Figure 46. — Semisynthesis of the KvAP voltage-dependent K+ channel using EPL and NCL.¹⁹²

In another example of semisynthesis as applied to a membrane protein, the Valiyaveetil laboratory used EPL to assemble the Na⁺-coupled aspartate transporter, GltPh.¹¹⁸ This 418-amino acid protein was assembled using an N-terminal protein α-thioester (residues 1-384) and a synthetic C-terminal 33-residue peptide. Folding of the semisynthetic protein afforded a transporter with a K_d value for aspartate binding similar to a native GltPh control.¹¹⁸ Substituting the highly conserved Arg397 residue in the aspartate binding pocket with the isosteric citrulline demonstrated that this residue is critical for Na⁺-coupled binding of aspartate and vesicular uptake of the amino acid in vitro.

7.3.2. G-Protein Coupled Receptors

GPCRs are the largest family of cell surface receptors in the human genome and are the targets of over 30% of current drugs on the market. Activated GPCRs are phosphorylated on their C-terminal cytosolic regions by GPCR kinases, a modification that leads to the binding of β-arrestin, triggering several downsteam processes including desensitization, internalization and signaling.^{559, 560} Consequently, access to phosphorylated versions of GPCRs could help further our understanding of these regulatory processes. With this in mind, Lefkowitz and coworkers employed sortase-mediated ligation to generate a hyperphosphorylated version of the β2-adrenergic receptor (β₂AR).⁵⁶¹ An evolved sortase⁴³⁰ was used to ligate a synthetic polyphosphorylated peptide to a recombinant version of the receptor equipped with a C-terminal LPETG sortase recognition sequence (Figure 47a). The semisynthetic protein was then reconstituted into high-density lipoprotein particles (nanodiscs).⁵⁶² Significantly, polyphosphorylation of the GPCR led to recruitment of β-arrestin, allosterically enhancing the affinity of cyanopindolol, an inverse agonist, for the receptor.

In related work, Shimada and coworkers performed NMR analysis on a segmentally labeled β₂AR prepared in vitro using PTS (Figure 47b).⁵⁶³ To observe only resonances corresponding to the C-terminus of the receptor, the authors expressed a C-terminally truncated form of the protein (residues 1-348) in insect cells, fused to the Int^N fragment of the Npu DnaE split intein. The remainder of β₂AR was expressed in E. coli (in ¹³C/¹⁵N containing media) fused to the Int^C fragment, and subsequently spliced onto the β₂AR-core. The segmentally labeled ligation product was then reconstituted into nanodiscs, and analyzed by NMR spectroscopy in unmodified form or phosphorylated by GRK2. Interestingly, the receptor C-terminus showed conformational changes upon phosphorylation, potentially caused by differential engagement with the transmembrane region of the GPCR.

7.4. Signaling Proteins

Cellular signaling events require complex networks of signal transduction pathways to interpret and respond to extracellular stimuli. These pathways are typically composed of multiple highly choreographed protein-protein interactions, often driven by post-translational modifications such as protein phosphorylation, that connect extracellular stimuli to a transcriptional output. In this section we will discuss how protein semisynthesis has been applied to proteins involved in cell signaling and signal transduction, including secreted signaling proteins, scaffold proteins, small GTPases and transcription factors.

7.4.1. Secreted Signaling Proteins

Cells secrete a wide range of small proteins, including those historically classed as growth factors, cytokines and hormones, in order to communicate among one another. These couriers of biological messages carry out their biological assignment through engagement with dedicated transmembrane receptors on target cells, which in turn initiate intracellular signaling cascades terminating in a transcriptional output. The activity of these secreted messenger proteins is tuned through extensive modifications that occur during passage through the endoplasmic reticulum and golgi apparatus. Protein chemistry has been used to access these modified proteins, aided by their (typically) small size and ability to be refolded in vitro.

The class of chemotactic cytokines known as chemokines have been accessed using both totally synthetic^{13, 564–573} or semisynthetic^{132–139, 188, 574, 575} protein chemistry. Typically, these proteins are only 70-80 residues and have conserved cysteine residues that are conveniently located for synthetic manipulation. Indeed, the chemokine CXCL8, also known as interleukin 8 (IL-8), was the first protein to be prepared using the native chemical ligation strategy.¹³ EPL has also been used extensively in the preparation of semisynthetic forms of two chemokines, CXCL8^{132–136, 574} and CXCL12^{137–139, 575} (stromal cell-derived factor 1). Beck-Sickinger and coworkers have developed robust semisynthetic routes to these chemokines (Figure 48a), allowing the introduction of fluorescent probes,^{133, 139} photocrosslinking groups,¹³⁴ photocages,^{138, 139} isotope labels,¹³⁶ and β-peptide mimics of the CXCL8 C-terminal helix.¹³⁵ In this last example, semisynthetic CXCL8 containing an oligo β³-amino acid sequence was prepared, projecting the native functionalities from an artificial secondary structure element in an otherwise native protein scaffold (Figure 48b).¹³⁵ Remarkably, these chimeric molecules retained activity in CXCR1 stimulation assays.

Figure 48. — Semisynthesis of chemokines. a) General strategy for the semisynthesis of modified CXCL8 and CXCL12, and examples of modifications incorporated. b) Semisynthesis of CXCL8 with a β-peptide sequence at the C-terminus.¹³⁵

Interleukins are immunomodulatory cytokines, and have provided an arena for the development and implementation of semisynthetic protein chemistry as applied to glycoproteins.^{187, 188, 576} The heterogeneity of protein glycosylation in vivo makes chemical preparation of such proteins with chemically defined glycans an attractive option with the added appeal of conquering such synthetically demanding molecules, even through total synthesis^{577, 578}. Unverzagt and coworkers used an impressive three-piece ligation strategy to assemble interleukin 6 (IL-6) with an N-linked nonasaccharide.¹⁸⁷ N- and C-terminal fragments were expressed recombinantly, the former using a tripartite fusion with two contiguous inteins (Ssp DnaB and Mxe GyrA), each undergoing autoprocessing to liberate the desired IL-6 fragment as an α-thioester with a free N-terminus (Figure 49). Extensive chemical expertise was required to troubleshoot and optimize the synthesis, including the efficient access to the synthetic central fragment, a hexapeptide α-thioester/hydrazide bearing the N-linked nonasaccharide. Nonetheless, the campaign was successful, and gave access to glycoforms of IL-6, bearing N-linked mono- or nonasaccharides, that were correctly folded and were as potent as recombinantly expressed IL-6 in stimulating the proliferation of IL-6 responsive pro B cells (Ba/F3).^{187, 579} To prepare a glycosylated form of interleukin 13 (IL-13), Kajihara and coworkers used a four-fragment assembly, including two central synthetic peptides and two flanking recombinant fragments (Figure 50).¹⁸⁸ The authors used an innovative method to access both N- and C-terminal fragments from a single recombinant protein, employing a combination of chemical cleavage events, including cyanogen bromide cleavage and the so-called “guanidine method”¹⁸⁸ of activation and transformation of cysteinyl-peptides into α-acylguanidines – α-thioester equivalents. Ultimately, the authors were able to assemble the 112-amino acid cytokine, bearing an N-linked nonasaccharide at Asn52.

Figure 49. — Semisynthesis of glycosylated interleukin 6.¹⁸⁷

Figure 50. — Semisynthesis of glycosylated interleukin 13.¹⁸⁸

Of all the glycoproteins studied in the biomedical fields, none have attracted the attention of synthetic protein chemists like erythropoietin (EPO), a hormone that stimulates the production of red blood cells.^{580, 581} Much of the gravitation pull of this 166-residue glycoprotein stems from its proven medical value – the recombinant version of the protein is used clinically in the treatment of anemia. EPO is natively N- and O-glycosylated at four positions, modifications which are critical for prolonged circulation in vivo,⁵⁸² although this glycosylation is heterogeneous. This factor opened the door to protein chemistry as a path to homogeneous EPO proteins, using both totally synthetic^{331, 583–587} and semisynthetic^144–146 strategies, to then be used to understand the effects of the carbohydrates on EPO’s pharmacokinetics. Collectively, these studies showcase the incredible power and versatility of modern protein and carbohydrate chemistries – the recent disclosure by the Kajihara group of the total synthesis of five EPO glycoforms provides an especially stunning example of these capabilities.⁵⁸⁷ While the preparation of native glycoforms represents a major achievement, the considerable synthetic efforts associated with these synthetic campaigns has motivated researchers to consider neoglycoprotein surrogates in order to expedite downstream biological study. Macmillan and coworkers used NCL to prepare semisynthetic EPO with an alkynylated side-chain at position 24, a handle to install a glycan mimic using click chemistry,⁵⁷ from a recombinant C-terminal protein fragment and an N-terminal peptide α-thioester. Kajihara and coworkers used a similar neoglycoprotein strategy to prepare EPO with two¹⁴⁵ and three¹⁴⁴ complex sialyloligosaccharides. More recently, Rubini and coworkers reported an amber suppression strategy to site-specifically install a propargylated-lysine residue at various positions in fully recombinant EPO.⁵⁸⁸ Subsequent, click chemistry using N-glycan azides, yielded several glysosylated EPO conjugates that were shown to be more stable to aggregation relative to the non-glycosylated version of the protein. These and other syntheses of EPO have helped confirm the importance of glycosylation, in particular the degree of sialylation, for the activity of this protein hormone in vivo. More generally, these studies have broken through many of the technical barriers associated with gaining access to homogenous glycoproteins.

7.4.2. Scaffold Proteins

Scaffold proteins play important roles in cellular signaling, serving as hubs for the coordination of signaling cascades through the localization and activation of specific proteins.⁵⁸⁹ These scaffolds are structurally diverse, but are all typically composed of a modular assembly of discrete binding domains specific for certain motifs present within binding partners. The complex circuitry that these molecular adapters template, further regulated by modifications to the proteins themselves, have made the study of scaffold proteins an important area of investigation. The Crk family of adaptor proteins are involved in many cellular processes including proliferation. These proteins are composed of modular Src Homology 2 (SH2) and Src Homology 3 (SH3) domains that serve as docking sites for protein-protein interactions. Phosphorylation of Tyr221, by the kinases c-Abl and EGFR, leads to intramolecular association between this residue and the N-terminal SH2 domain, forming a closed autoinhibited conformation. These conformational changes have been observed on semisynthetic Crk-II using FRET- and NMR-based methods.^{108, 109, 130, 590, 591} Strategic placement of donor and acceptor fluorophores onto the N- and C-termini of a Crk-II protein afforded a fluorescent reporter for phosphorylation at Tyr221, providing a direct readout of Abl kinase activity.^{130, 590} Using sequential protein ligations, the individual domains of Crk-II were also assembled¹⁰⁹ is a fashion allowing NMR analysis of the modular protein through segmental isotopic labelling.^{364, 592} Indeed, NMR-analysis of the segmentally labelled C-terminal SH3 domain of Crk-II, helped to provide a model for Crk-II activation and autoinhibition, where destabilizing structural deformation of this domain counteracts the stabilizing interdomain interactions.¹⁰⁸

Protein semisynthesis has been used to study the postsynaptic density protein 95 (PSD-95), an abundant scaffolding protein in the post synaptic density (PSD), a dense network of proteins at the membrane of post synaptic neurons.⁵⁹³ PSD-95 contains three PDZ domains that mediate protein-protein interactions with the C-termini of a range of proteins including neuronal receptors, interactions that concentrate proteins to the PSD.⁵⁹³ In order to understand how these modules engage their ligands, Stromgaard and coworkers used both NCL and EPL to generate semisynthetic forms of the three PDZ domains of PSD-95, bearing either phosphorylation²³¹ or backbone amide-to-ester²²⁹ chemical modifications (Figure 51). Phosphorylation of four residues throughout the three PDZ domains (S73ph, Y236ph, Y240ph and Y397ph) generally led to decreased ligand binding, with the exception of stargazin, an auxiliary subunit of the APMA receptor, which displayed increased binding to PDZ3 (Y397ph). This result points to finer regulation of this interaction through post-translational modification of both PSD-95 and stargazin.⁵⁹⁴ Semisynthetic PDZ domains bearing amide-to-ester mutations within the conserved carboxylate-binding-site showed decreased binding affinity for PDZ-ligands, confirming the importance of H-bonding interactions in the selectivity of binding partners.²²⁹ This work has contributed greatly to our understanding of PDZ-ligand engagement generally, and has helped fuel ongoing research into the development of pharmacological agents that disrupt PDZ-ligand interactions for the treatment of various diseases.⁵⁹⁵

Figure 51. — Semisynthesis of PSD-95 PDZ domains. a) Semisyntheses of phosphorylated PDZ1–3. b) Semisynthesis of PSD-95 PDZ2 with amide-to-ester mutants at the substrate binding site. Right: Important H-bonding interactions in the PDZ-substrate complex backbone, mapped onto a canonical PDZ-substrate structure (PDB ID: 1BE9).²²⁹

7.4.3. Small GTPases

Small GTPases are a family of small GTP-binding proteins that play critical roles in the cell, including signal transduction, nuclear import/export, and membrane trafficking. Semisynthesis has been extensively used in the preparation of Rab^{241–244, 319, 320} and Ras^248–250 GTPase proteins in C-terminally prenylated forms. This modification is necessary for association of this soluble protein to the cytoplasmic leaflet of the plasma membrane.^{596, 597} In early work in this area, Alexandrov and coworkers used EPL to generate a semisynthetic Rab GTPase containing a C-terminal fluorophore and used this to study the interaction with a Rab geranylgeranyl transferase.²⁴⁰ This group further developed their semisynthetic approach in order to chemically install a C-terminal prenyl modification onto Rab7 (Figure 52), using EPL to ligate a recombinant Rab7 α-thioester to a C-terminally prenylated synthetic peptide.²⁴⁴ This basic strategy was subsequently applied to the semisynthesis of the Rab GTPase Ypt1, both in monoprenylated³¹⁹ and diprenylated³²⁰ forms. Impressively, both the singly and doubly prenylated Ypt1 proteins were crystallized in complex with the Rab GDP-dissociation inhibitor protein (GDI), which is responsible for the extraction of GDP-bound Rabs from membranes.^{319, 320} These structures revealed the binding modes of the two prenyl groups on the Ypt1 C-terminus, one buried in a hydrophobic cavity of GDI and the second laying adjacent to this cavity on the surface of the chaperone protein.³²⁰ The binding strength between GDI and prenylated Rab proteins have been subsequently measured in vitro, revealing that both mono and diprenylated GDP-bound Rab7 are bound to GDI with high affinity (K_d 1-5 nM), whereas unprenylated Rab7 is bound weakly (K_d >50 uM).²⁴³ However, this high affinity interaction is abolished when the Rab7 protein is bound to a GTP-analog.²⁴¹ These structural and biochemical experiments have provided key insights into how the Rab:GDI interaction is regulated, helping shape a model for how Rab GTPases are targeted to membranes.

Figure 52. — Semisynthesis of prenylated Rab GTPases. a) Scheme for the semisynthesis of prenylated Rab7 GTPase.²⁴⁴ b) Co-crystal structure of the semisynthetic doubly prenylated Rab GTPase Ypt1 (green cartoon) and GDP dissociation inhibitor (GDI, white surface).³²⁰ Unstructured C-terminus is shown as dashed green line. Expansion shows a zoomed view of the two geranylgeranyl moieties. (PDB ID: 2BCG)

7.4.4. TGF-β signaling pathway

Protein semisynthesis has helped our understanding of discrete steps in transforming growth factor-β (TGF-β) signaling, a pathway involved in many processes including cell proliferation, differentiation and migration.^{598, 599} TGF-β receptors are heterodimeric transmembrane serine/threonine kinases composed of type I and type II TGF-β receptor subunits (TBR-I and TBR-II) that undergo transphosphorylation upon ligand binding. Phosphorylation of TBR-I leads to the activation of this receptor kinase and phosphorylation of a set of transcription factors known as R-Smads. Semisynthesis of the cytoplasmic region of TBR-I illuminated how the receptor becomes activated upon phosphorylation.^{307, 308, 600} Using NCL, a tetraphosphorylated form of TBR-1 was generated semisynthetically, requiring a challenging synthesis of a peptide α-thioester bearing four phosphorylated serine and threonine residues.^{307, 308} Through access to these proteins, it was found that phosphorylation of TBR-I plays a duel role in the activation of the receptor, by simultaneously decreasing the affinity for the inhibitory protein FKBP12 as well as increasing the affinity for a substrate R-Smad protein (Smad2).⁶⁰⁰ Subsequently, semisynthesis of Smad2 itself was undertaken in order to understand the structural effects of C-terminal phosphorylation on this transcription factor.²⁶⁷ Indeed, semisynthetic Smad-2 formed heterotrimers in a phosphorylation dependent manner (Figure 53a). X-ray crystallography revealed this conformational switch is driven by the interaction the phosphoserine residues on the C-terminus of one Smad2 molecule and the mad homology 2 (MH2) domain of a separate molecule.²⁶⁷ Further analysis of phosphorylated variants of Smad2 indicated that both phosphorylation events were required for stable formation of this homotrimer.²⁶⁸ This interaction was captured through covalent UV-crosslinking through a synthetically installed diazirine-containing amino acid.²⁶⁹ Additionally, the incorporation of UV-labile ‘photocages’ onto these key phosphates afforded temporal control over this association, blocking trimerization until deprotected through UV irradiation (Figure 53b).²⁷⁰ Note that in an extension of this caging idea, a semisynthetic version of Smad2 was generated that allowed simultaneous triggering of activity and fluorescence, allowing for tracking of the active protein in live cells.^{271, 272} The insights gained from these various studies have proved to be significant for the molecular level understanding of modification-driven regulatory events along the entire TGF-β signaling pathway.

Figure 53. — Control of Smad2 activity using semisynthesis. a) Association of semisynthetic phosphorylated Smad2 into trimers (PDB ID code 1KHX). b) Photocaging of Smad2 association and activation.²⁷⁰

7.5. Enzymes

Enzymes have been targets of protein semisynthesis (and, of course, total synthesis) since the earliest days.^{97, 465, 601–603} The intricacies of enzyme catalysis coupled with the complex modes of allosteric regulation Nature employs to control activity, including through PTMs, means that standard mutagenesis is sometimes a rather blunt instrument when it comes to mechanistic dissection. Semisynthesis, by contrast, offers far greater flexibility in terms of what can be incorporated into an enzyme active site, in principle, making it a more precise tool. In this section, we illustrate this point using select examples from what is a very active area.

7.5.1. Ribonuclease A

Ribonuclease A (RNase A) is a particularly resilient enzyme, and has thus served as a valuable model for the use of protein chemistry to study enzyme structure and function.⁶⁰⁴ Of note, RNase A was first synthesized in 1969 via two routes, stepwise solid-phase peptide synthesis by Gutte and Merrifield,^{601, 602} and convergent synthesis by Hirschmann and coworkers.⁶⁰³ Indeed, RNase A has consistently featured in landmark studies in the peptide and protein chemistry fields; the reader is reminded that the enzyme was assembled from a series of synthetic peptide fragments using subtiligase-mediated ligation (see section 5.1). RNase A was also one of the first proteins to be accessed using intein chemistry, assembled semisynthetically by Xu and coworkers as a means to access the cytotoxic enzyme from catalytically inactive recombinant and synthetic fragments (Figure 54a).⁹⁸ Raines and coworkers used a similar strategy to incorporate a selenocysteine at position 110 using Sec-mediated EPL.²⁵¹ In both cases, semisynthetic RNase A had equivalent activity to the wild type enzyme, showing that these enzymes were properly folded even when containing a Cys110Sec mutation. Extending the ligation junction to Cys95, Raines and coworkers installed dipeptidyl turn mimics,^{252, 253} based on the β-amino acids 1 (Nip) or homoalanine (hAla), in order to study the folding and stability of RNase A (Figure 54b). While both of these backbone mutants displayed unchanged enzymatic activity relative to the WT enzyme, the conformational stabilities were affected, negatively in the case of the β-hAla modules but positively in the case of the more rigid Nip-based turns. Subsequent NMR analysis of semisynthetic RNase A, labeled with ¹⁵N an ¹³C isotopes exclusively at Pro-114, illuminated a cis-amide conformational preference at this position.⁶⁰⁵ The replacement of Pro-114 with a 5,5-dimethylproline (dmPro) residue afforded RNase A with enhanced conformational stability, due to the increased propensity to adopt a cis-peptide bond (Figure 54b).²⁵⁶ Variants containing 1,5-triazole mimics of Pro-114 were also able to support this conformation.²⁵⁴ Interestingly, replacement of Pro-114 with (2S,4S)-4-fluoroproline (a single atom replacement) can even promote this conformational preference through a weakened n→π* interaction, which ordinarily favors the trans isomer.²⁵⁵ Unverzagt and coworkers developed a semisynthetic route to RNase bearing a complex N-linked glycan at Asn34.^{257, 258} Initial plans to assemble the protein from a recombinant C-terminal fragment and a synthetic peptide bearing a large biantennary glycan were stymied by inefficient peptide synthesis caused by the large nonasaccharide moiety. This necessitated a three-piece ligation strategy between two synthetic peptides (comprising residues 1-25 and 26-39) and a recombinantly expressed fragment from residues 40-124. As is the case for all synthetic preparations of homogeneous glycoproteins, this successful semisynthesis of glycosylated RNase is a tour-de-force, requiring the integration of many tools of carbohydrate, peptide and protein chemistry.

Figure 54. — Semisynthesis of Ribonuclease A (RNase A). a) Scheme for the semisyntheses of RNase A using EPL at Cys or Sec.^{98, 251} b) Dipeptidyl turn mimics installed in RNase A using EPL. Melting temperatures from themally induced unfolding experiments are shown.

7.5.2. Protein Kinases

Understanding how protein kinase activity is regulated has been an intense area of research for decades, spurred on by the intimate relationship between abnormal signaling and cancer.⁶⁰⁶ The first report of EPL involved the semisynthesis of a phosphorylated form of the tyrosine kinase Csk, a non-receptor tyrosine kinase that phosphorylates the tail of the Src family of kinases.⁹⁷ Since this pioneering work, protein semisynthesis has been used to access various kinase enzymes (Table 1), including the TGF-β receptor kinase (also see section 7.4.4),^{307, 308, 600} Eph tyrosine kinases,^{142, 143} protein kinase A,²³⁹ Rad53,²⁴⁵ p38α MAPK,²²³ casein kinase II,¹²⁴ Src,²⁷⁵ and PDK1.⁶⁰⁷ The kind of questions that these studies have helped address is well illustrated by the work of Cole and coworkers on casein kinase II.¹²⁴ In this case, EPL was used to install two different modifications into the protein, phosphothreonine at position 344 and O-GlcNAc at Ser347. While the modified proteins showed little to no difference in terms of kinase activity for a substrate peptide, assays on human protein microarrays showed that kinase substrate specificity can be modulated by these two PTMs. Additionally, Thr344 phosphorylation leads to increased cellular stability of casein kinase II, as assessed by microinjection of a semisynthetic protein containing a non-hydrolyzable PTM mimic into cells.¹²⁴

Protein semisynthesis has illuminated distinct mechanisms of Akt1 kinase activation by phosphorylation.¹¹⁰ Site-specifically phosphorylated Akt1 kinases were prepared through EPL employing recombinant Akt1 α-thioesters (residues 1-459) and synthetic phosphopeptides modified at Ser473, Ser477 and/or Thr479 (residues 460-480) (Figure 55). Fortuitously, the native residue of Akt1 at position 460 is Cys, allowing a traceless semisynthesis to be achieved without the need for any desulfurization steps. In order to install phosphorylation on Thr308, which resides within the kinase activation loop, the Akt1 α-thioester fragment was treated with the protein kinase PDK1. Thus, the final semisynthetic proteins containing both enzymatically and chemically installed phosphorylated amino acids. The authors went on to determine that activation of kinase activity by phosphorylation at Ser473 operates through interaction with the linker region between the kinase domain and the N-terminal pleckstrin homology (PH) domain, thereby relieving autoinhibition.¹¹⁰ Conversely, activation by Ser477/Thr479 phosphorylation appears to function through direct interaction with the activation loop that reduces autoinhibition by the PH domain.

Figure 55. — Semisynthesis of Akt1 kinase. a) Scheme for the semisynthesis of phosphorylated Akt1 kinase using EPL.¹¹⁰ b) Model for the modulation of Akt1 activity through phosphorylation.

7.5.3. Phosphatase enzymes

Protein phosphatase enzymes can themselves be regulated by phosphorylation. Protein semisynthesis has allowed these regulatory mechanisms to be revealed. Cole and coworkers used EPL in order to understand how tyrosine phosphorylation of SHP-2 can affect the activity of this protein tyrosine phosphatase.^{265, 266} Due to the tendency to autodephosphorylate, the authors prepared semisynthetic SHP-2 containing a non-hydrolyzable phosphotyrosine analogue through EPL between a recombinant protein α-thioester and synthetic peptides containing either phosphonomethylene-phenylalanine or difluorophosphonomethylene-phenylalanine residues in place of phosphotyrosines (Figure 56a). The authors were able to demonstrate that these phosphonotyrosine modifications stimulated the phosphatase activity of SHP-2 through intramolecular interactions with the SH2 domains (Figure 56b). Further, microinjection of semisynthetic SHP-2 bearing phosphonotyrosine at Tyr542 into cells showed that this protein was able to stimulate the MAP kinase pathway more potently than the unmodified protein.

Figure 56. — Semisyntheses of SHP-2 phosphatase. a) Scheme for the semisynthesis of SHP-2 using EPL.^{265, 266} b) Model for the activation of SHP-2 phosphatase activity through tyrosine phosphorylation.

Originally thought to be a protein tyrosine phosphatase, the phosphatase and tensin homolog (PTEN) is a lipid phosphatase that is regulated by multiple PTMs, including phosphorylation of C-terminal Ser and Thr residues. Semisynthetic PTEN has been generated in order to understand how these modifications affect protein activity.^{226–228, 471} An initial semisynthesis of the protein involved EPL between a recombinantly produced PTEN α-thioester (residues 1-378) and a tetraphosphorylated synthetic peptide (residues 379-403).²²⁸ Notably, mutation of the native Tyr379 to Cys residue was necessary for the ligation reaction, a mutation that has since been shown not to be silent.⁴⁸⁴ As noted in section 6.3, this issue was subsequently overcome via subtiligase-templated ligation between a PTEN α-thioester (residues 1-377) and a tetraphosphorylated synthetic peptide (residues 378-403) bearing an N-terminal Arg residue (Figure 57a).⁴⁷¹ The phosphorylated PTEN proteins displayed reduced catalytic efficiency relative to the unmodified protein, as well as reduced affinity for lipid vesicles.^{228, 471} Phosphorylation of the C-terminus was shown to promote a more compact form of PTEN, through interactions between the phosphorylated tail and the core that were identified through photocrosslinking and mass-spectrometry (Figure 57b).²²⁶ This compact phosphorylated form of PTEN was subsequently demonstrated to be a poorer substrate for ubiquitination by the E3 ligase WWP2,⁶⁰⁸ suggesting that phosphorylation both regulates the activity and the stability of PTEN. Of note, WWP2 is itself regulated by phosphorylation of an autoinhibitory linker region, a mechanism also illuminated through protein semisynthesis.²¹⁶

Figure 57. — Semisyntheses of PTEN. a) Scheme for the semisynthesis of phosphorylated PTEN using subtiligase-catalyzed EPL.⁴⁷¹ b) Model for the modulation of PTEN structure through phosphorylation.²²⁸

7.5.4. p300

The transcriptional coactivator p300 is a histone acetyltransferase (HAT) that is critical for regulating gene expression in mammalian cells.⁶⁰⁹ Efforts to recombinantly express the p300 HAT domain in E. coli were hampered by autoacetylation of the protein in vivo, as well as the potential to indiscriminately acetylate E. coli host proteins.²²¹ Cole and coworkers found that this protein could be accessed efficiently through semisynthetic assembly of a recombinant α-thioester (spanning residues 1287-1652 of the full length protein), and a 14 amino acid synthetic peptide.^{221, 222, 610, 611} Importantly, the C-terminally truncated sequence was almost devoid of catalytic activity compared to the semisynthetic product, allowing the authors to study the hypoacetylated form of the enzyme.²²¹ Interestingly, catalysis by the semisynthetic enzyme was stimulated after allowing for autoacetylation to take place. Acetylated residues important for p300 autoactivation were found to reside on a proteolytically sensitive loop region thought to autoinhibit p300 HAT activity. Further studies demonstrated that autoacetylation primarily occurs in trans on up to 17 lysine residues.⁶¹² To assess the contributions of individual lysine acetylation events to p300 activity, Cole and coworkers engineered a circularly permuted form of the protein (cp-p300), relocating the autoactivation loop from the middle of the HAT domain to the C-terminus, which could then be modified semisynthetically (Figure 58).²²² A series of synthetic 38-mer peptides containing the autoactivation loop and modified with varying numbers of acetyllysine residues, were ligated to a recombinant α-thioester containing remainder of the cp-p300 domain. Under conditions where autoacetylation is minimal, these semisynthetic proteins displayed increasing levels of HAT activity with increasing loop acetylation. Indeed, the cp-p300 domain with 6 preinstalled acetylation sites was equally active as the p300 HAT domain with this loop deleted. Not only do these studies shed light on the regulation of this important coactivator enzyme, they suggest an alternative way to study the internal region of a protein through semisynthesis of a circularly permuted analogue.

Figure 58. — Circular permutation of the p300 HAT domain (PDB ID: 3BIY), which was accessed semisynthetically in order to install acetylated lysine residues.²²²

7.5.5. Serotonin N-Acetyltransferase

Serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AANAT)] is a key regulator of circadian rhythms, responsible for the conversion of serotonin into N-acetyl serotonin, the precursor to melatonin. Phosphorylation of AANAT by PKA at positions Thr31 and Ser205 leads to stabilization of the enzyme, a mechanism that had been presumed to occur through enhanced interaction with two sites of the 14-3-3 protein. This interaction was studied using semisynthetic AANAT, bearing modifications at Thr31 and/or Ser205 as phosphates or the non-hydrolyzable analogues phosphonomethylenealanine (Pma) and phosphonodifluoromethylenealanine (Pfa).^{116, 117} Pulldown experiments showed that AANAT modified at these positions (Thr31ph/Pma or Ser205ph/Pfa) showed greater affinity for the 14-3-3 protein in vitro than unmodified AANAT, or a Thr31Glu phospho-mimic. Furthermore, these two modifications were able to confer stability to AANAT that was microinjected into cells. Interestingly, phosphorylation at Thr31 was shown to activate the catalytic activity of AANAT through an interaction with 14-3-3ζ that decreases the Km for the substrate tryptamine, although this activation is abolished in the case of the dually modified protein.¹¹⁵ Notably, this enzyme activation was not observed using a serine-to-glutamate mutation as a mimic of phosphoserine, a strategy often applied in cell biology experiments. Analogous results in other systems highlight important limitations of this mutagenesis strategy to study protein phosphorylation, specifically for cases where molecular recognition is strongly dependent on the geometry and charge state of the of phosphoserine/threonine side chains, properties which are often poorly simulated by the glutamate side chain carboxylate.⁶¹³

7.5.6. Sortase A

Protein chemistry has been used to study the structure and activity of sortase A, itself a valuable tool for protein chemistry. Indeed, refolded totally synthetic forms of the catalytic domain of sortase A are at least as active as the recombinant protein.⁶¹⁴ In an attempt to resolve conflicting views regarding the role of the conserved basic Arg197 residue in transpeptidation by the enzyme, McCafferty and coworkers used semisynthesis to incorporate the isosteric and non-ionizable citrulline residue at this position (Figure 59a).²⁷⁴ In this case, a sortase A (25-183) α-thioester was ligated to a synthetic peptide comprising residues 184-206, bearing an N-terminal cysteine and a citrulline at position 197. The citrulline mutation resulted in only modest decrease (less than 3-fold) in catalytic efficiency of the enzyme, arguing for a model of transition-state stabilization through hydrogen-bonding rather than electrostatic interactions mediated by the Arg side-chain (Figure 59b). Schwarzer and coworkers used a semisynthetic approach in order to probe the effects of mutating the catalytic cysteine residue (the only cysteine present in the transpeptidase domain) to either homocysteine or selenocysteine.²⁷³ Interestingly, the pH-profile of the Sec-sortase was shifted towards more acidic conditions, although this enzyme displayed a 2-3-fold reduction in activity relative to the WT sequence.

Figure 59. — Semisynthesis of the catalytic domain of sortase A (SrtA). a) Scheme for the semisynthesis of citrullinated SrtA using EPL.²⁷⁴ b) Molecular mechanism of transpeptidation facilitated by H-bonding between the sorting motif and Arg/Cit.

7.5.7. Inteins

As is hopefully clear by this point, the field of protein chemistry has benefitted greatly from exploiting the protein splicing activity of inteins. In turn, these enabling technologies have fed forward into the study of inteins themselves, providing a better understanding of the autocatalyzed protein splicing mechanism. In an early example of this, NCL was used to study the first step in the splicing cascade of the Mxe GyrA intein, namely the N-to-S acyl shift at the N-terminal splice junction (see Figure 10). Specifically, NMR active ¹⁵N and ¹³C isotopes were selectively incorporated at the scissile amide bond by ligating a synthetic peptide α-thioester, containing a single ¹³C atom at the C’ position of the C-terminal residue, to a uniformly ¹⁵N-labeled version of the intein.²¹² Analysis of the one-bond dipolar coupling between these atoms indicated that this scissile amide is significantly distorted in a manner dependent on the catalytic His75 residue, supporting a role for ground-state destabilization in the initiation of the splicing cascade.

Semisynthesis of the Mxe GyrA protein has also provided insight into the penultimate step of protein splicing, the resolution of the branched intermediate through succinimide formation.^{213, 214} To generate this branched protein, a recombinant α-thioester fragment corresponding to residues 1-184 of the Mxe GyrA intein was ligated to a synthetic peptide containing the native N- and C- exteins branched through the side-chain of the catalytic +1Thr residue (Figure 60a). Ironically, this semisynthesis actually employed an intein to make an intein (i.e to install the α-thioester into the Mxe GyrA fragment). The refolded semisynthetic, branched intermediate was found to undergo C-terminal asparagine cyclization an order of magnitude faster than a linear intein unable to form the branched intermediate due to a C1A mutation. Additionally, the necessity of histidines 187 and 197 as catalysts for the asparagine cyclization reaction was established through the replacement of these residues with isosteric β-thienylalanines. This insight allowed the x-ray crystal structure of a ‘trapped’ semisynthetic branched intermediate to be solved,²¹⁴ revealing subtle rearrangements of amino acid side-chains in the active site, such that the C-terminal asparagine residue is positioned for nucleophilic attack on the +1 amide bond, further activated by hydrogen-bonding to a backbone amide bond (Figure 60b).

Figure 60. — Semisynthesis of inteins. a) Scheme for the semisynthesis of the *Mxe* GyrA branched intermediate using EPL.²¹⁴ b) Close-up of the active site of the *Mxe* GyrA (extein residues are colored green and the intein is colored pink). Apparent H-bonds are indicated with dashed lines. c) Folding pathway for the *Npu* DnaE split intein.²¹⁹

The fragmented form of split inteins permits the study of catalytic mechanisms without the need to perform formal protein semisynthesis – the split intein complex can simply be constituted from synthetic and recombinant intein fragments.⁶¹⁵ However, protein semisynthesis has been used to study the structure of split intein fragments.²¹⁹ Using EPL, the 102-residue Int^N fragment of the Npu DnaE split intein was segmentally labeled with ¹⁵N isotopes for NMR spectroscopy. The intein fragment displayed a bipartite structure, where the N-terminal portion is partly folded while the remainder is unstructured in the absence of the cognate Int^C fragment. This insight proved instrumental in piecing together the overall folding pathway of Npu DnaE, which involves an initial electrostatic capture step, involving the disordered regions of the Int^N and Int^C fragments, followed by a hydrophobically driven collapse into the final native fold (Figure 60c). A notable feature of this folding mechanism is that it provides an explanation for why the isolated Int^N and Int^C fragments have no residual splicing activity, namely the catalytic residues are in disordered regions and only adopt a catalytic configuration upon fragment association. This basic paradigm is also true for the atypically split intein, Cat (Consensus atypical) TerL, even though the details of the folding pathway differ.³⁹²

8. SUMMARY AND FUTURE PERSPECTIVES

The marriage of chemical peptide synthesis and recombinant protein production, two vastly different methods of generating polypeptides, has provided controlled access to molecules that could not be generated otherwise. From an engineering point of view, it is quite remarkable that such large biomolecules can be stitched together in a test-tube, often without surgical scars that would give away any hint of non-biological origins. In assembling this review, our motives were two-fold: firstly, to provide an inspection of the available semisynthetic methods of targeted amide bond formation, and secondly to demonstrate the broad use of this enabling protein engineering strategy for the study of many classes of proteins.

The various semisynthetic methods discussed in this review range from those considered to be chemical in origin, such as NCL, to those that are more biologically inspired, including enzyme-catalyzed transpeptidation. These methods have often been considered separate, yet they are unified by common themes of selective acyl-shift chemistry to both break and make selective amide bonds. Enzymes and biocatalysts such as inteins, transpeptidases and proteases are evolved for such precision protein engineering in vivo and have been repurposed by protein chemists to also carry out these tasks in vitro. Currently, each semisynthesis method brings inherent strengths and weaknesses to a particular application, and no one strategy in its current form can truly carry out the role of an all-purpose protein assembly tool. Nonetheless, considerable progress continues to be made in broadening the scope of these strategies, whether it be through the development of new chemistries, the design or evolution of more agile protein ligases or the development of hybrid chemoenzymatic approaches that blend beneficial features of parental methods. Moreover, the field in general is likely to benefit from increased implementation of powerful advances in directed protein evolution.^{616, 617}

While it is remarkable just how far protein semisynthesis has come, both in terms of methodological advances and the biological questions that are now being addressed, it would be wrong to conclude that the field has matured to the point where any biological problem, any protein system, can now be reasonably approached. This is simply not the case. For example, most integral membrane proteins, despite their enormous biological importance, continue to be beyond the reach of current capabilities. Equally, many if not most proteins exist as part of large multi-subunit complexes, a situation that greatly complicates semisynthetic access to these systems, at least if one wishes to study the native complex. Thus, there are plenty of opportunities for profound methodological advancement in the field. Below, we discuss just two of these, recognizing that there are many others.

1. One-step semisynthesis applied to internal regions:

In theory, the size of a protein per se does not dictate whether it is an attractive target for semisynthesis through amide bond construction. Instead, it is the location of the desired modification site within the protein sequence that most often determines whether semisynthesis is a viable strategy. Semisynthetic targets where a modified fragment is installed at the N- or C-termini are the most facile, as these applications only require a single ligation to append a modified peptide onto the end of a truncated protein. On the other hand, modification of an internal region of a protein typically requires that a multistep ligation strategy (i.e. involving the assembly of three or more fragments) be adopted. While there are many impressive examples of this, several of which are described in this review, multistep semisyntheses can be technically quite demanding and are hence less appealing to the non-specialist. In principle, the ability to insert a polypeptide sequence within an expressed protein in a single step would lower the barrier to manipulating internal regions of a protein through semisynthesis. The use of orthogonal transpeptidase enzymes, for example butelase 1 and sortase A, could provide a solution to this problem, through the reversible metathesis of a recombinant sequence with a synthetic sequence through transpeptidation. Alternatively, one could also envision the use of PTS using orthogonal split inteins for such an application, where two templated splicing events irreversibly exchange the recombinant internal sequence with one derived synthetically. In this scenario, synthetic access to peptides bearing two split intein fragments would be required. Accordingly, these reactions would require the use of split inteins with atypically short fragments, or the in situ assembly of split intein fragments as used in TAIL.

2. Protein semisynthesis in the cellular milieu:

The frontier of protein semisynthesis appears, perhaps, not to be the refinement of existing technologies for in vitro applications. Instead, a true challenge is the literal transduction of these technologies into the cellular setting, where biological implications of biochemical and biophysical behavior become apparent. As mentioned previously, semisynthetic proteins may be delivered to cells, for example through microinjection, in order to observe the effects of a given chemical modification. However, in many cases it is preferable to reassemble the protein sequence within the cell itself, especially in cases where the protein target exists in larger complexes with other proteins or, in the case of histone proteins, genomic DNA. Accordingly, the vessel of the semisynthetic reaction is no longer the test tube, but the living cell. Somewhat cruelly, two of the principle tools for protein semisynthesis in vitro, NCL and EPL, are obstructed by the high concentration of reactive thiols in cellular environments. Biocatalytic methods, however, are viable alternatives, as they have been subject to evolutionary pressure in order to function on the surface of and inside living cells. Thankfully, tools capable of carrying out user-defined semisynthesis in cells are now becoming available, achieving a level of proficiency required for cellular applications. Among these, split inteins and transpeptidases have been used to modify proteins in living cells (as described earlier), and it is anticipated that use of these tools will be further explored to generate modified proteins in cells, presumably relying on advanced methods for the delivery of synthetic polypeptides across the plasma membrane, which we note is a very active area of study in chemical biology.^618–620 While the challenges associated of this undertaking can hardly be overstated, one could argue that these are no less daunting than those faced by the peptide chemistry community some thirty years ago when it considered protein semisynthesis. Thus, we feel there is some room of optimism here.

In conclusion, protein semisynthesis occupies a unique methodological space at the intersection of synthetic chemistry and molecular biology and has been fortunate enough to benefit from advances in both fields. The range of biological questions answered using semisynthetic proteins is testament to this synergy. This strategy will continue to provide innovative and accessible ways to modify the structure of proteins for molecular-level interrogation, driven by the constant appearance of challenging biological questions and the need to address these in a physiologic context.

ACKNOWLEDGMENT

We thank The Charles H. Revson Foundation (R.E.T.) and the National Institutes of Health (NIH, R37 GM086868 and P01 CA196539) for financial support. We also thank members of the Muir lab for helpful discussions.

ABBREVIATIONS

AA, aa: amino acid
A, Ala: alanine
C, Cys: cysteine
D, Asp: aspartic acid
E, Glu: glutamic acid
F, Phe: phenylalanine
G, Gly: glycine
H, His: histidine
I, Ile: isoleucine
K, Lys: lysine
L, Leu: leucine
M, Met: methionine
N, Asn: asparagine
P, Pro: proline
Q, Gln: glutamine
R, Arg: arginine
S, Ser: serine
T, Thr: threonine
U, Sec: selenocysteine
V, Val: valine
W, Trp: tryptophan
X, Xaa: any amino acid
Yaa: any amino acid
AANAT: arylalkylamine N-acetyltransferase, also known as serotonin N-acetyl transferase
AceL TerL: atypically split intein in the T4-bacteriophage-type DNA-packaging terminase from the Ace Lake in Antarctica
Acm: acetamidomethyl
ADP: adenosine diphosphate
Akt1: Akt serine/threonine kinase 1, also known as protein kinase B
AP-1: activator protein 1
APMA: μ-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
Ava DnaE: naturally split intein in the DnaE protein from Anabaena variabilis
b₂AR: b2-adrenergic receptor
Bn: benzyl
Boc: tert-butoxycarbonyl
Cas9: CRISPR associated protein 9
Cfa DnaE: consensus designed split intein from the fast-splicing DnaE split inteins
Cfa_GEP DnaE: Cfa DnaE with a loop mutation conferring increased extein tolerance
Chd1: chromodomain-helicase-DNA-binding protein 1, chromatin remodeler, component of the SAGA complex
CHO-K1: subclone of chinese hampster ovary cell line
CoREST: RE1 silencing transcription factor, a transcriptional corepressor
cp-p300: circularly permutated p300 HAT domain
CRISPR: clustered regularly interspaced short palindromic repeats
Csk: C-terminal Src kinase
CXCL12: chemokine (C-X-C motif) ligand 12, also known as stromal cell-derived factor 1
CXCL8: chemokine (C-X-C motif) ligand 8, also known as interleukin 8
Dbz: diaminobenzoyl
dCas9: nuclease dead mutant of Cas9
dmPro: 5,5-dimethylproline
DnaE: catalytic subunit of bacterial DNA polymerase III
Dot1: disruptor of telomeric silencing, H3K79 methyltransferase
DTA: diptheria toxin a-chain
EDTA: Ethylenediaminetetraacetate
EED: embryonic ectoderm development, member of the PRC2 complex
EGFR: epidermal growth factor receptor
EPL: expressed protein ligation
EPO: erythropoietin
eSrtA(2A): evolved SaSrtA recognizing LAxTG sorting sequence
eSrtA(4S): evolved SaSrtA recognizing LPxSG sorting sequence
ExtC: C-terminal extein sequence
ExtN: N-terminal extein sequence
EZH2: enhancer of zeste homolog 2, member of the PRC2 complex
F40: evolved SaSrtA recognizing yPxTG sorting sequences
FKBP12: FK506 binding protein 12
Fmoc: 9-fluorenylmethoxycarbonyl
FRET: Förster resonance energy transfer
GDI: GDP-dissociation inhibitor protein
GFP: green fluorescent protein
GlcNAc: N-acetyl glucosamine
GltPh: Na⁺-coupled aspartate transporter
GOS TerL: atypically split intein in the T4-bacteriophage-type DNA-packaging terminase from the Punta Cormorant lagoon in the Galapagos
gp41-1/8: split inteins found in the gp41 DNA helicase from unknown host
GPCR: G-protein coupled receptors
GPI: glycosylphosphatidylinositol
GRK2: G-protein coupled receptor kinase 2
hAla: homoalanine
HAT: histone acetyltransferase
HDAC: histone deacetylase
HED: homing endonuclease domain
HEK293T: Human embryonic kidney 293 cell line expressing a mutant version of the SV40 large T antigen
HF: hydrogen fluoride
hGH: human growth hormone
HP1a: heterochromatin protein 1a, binds H3K9me2/3
IL-13: interleukin 13
IL-6: interleukin 6
IMPDH-1: split intein found in the inosine-5’-monophosphate dehydrogenase from unknown host
Int^C: C-terminal split intein fragment
Int^N: N-terminal split intein fragment
ISWI: imitation switch, chromatin remodeler family
Jarid2: jumonji and AT-rich interaction domain containing 2, member of the PRC2 complex
KcsA: potassium channel from the soil bacteria Streptomyces lividans
KvAP: voltage-dependent potassium channel from Aeropyrum pernix
LC3: microtubule-associated protein light chain 3
LF_N: lethal factor N-terminal domain from anthrax toxin
MBP: maltose-binding protein
MeBn: methylbenzyl
MeCP2: methyl-CpG binding protein 2
MeOBn: methoxylbenzyl
MESNa: mercaptoethanesulfonate sodium salt
MH2: mad homology 2
MLL: myeloid/lymphoid or mixed-lineage leukemia, H3K4-specific methyltransferase
Mtu RecA: intein in the DNA repair protein RecA of Mycobacterium tuberculosis
Mxe GyrA: intein in DNA gyrase A protien of Mycobacterium xenopi
NCL: native chemical ligation
Nip: nipecotic acid
Npu DnaE: naturally split intein in the DnaE protein from Nostoc punctiforme
NrdJ-1: split intein found in the ribonucleotide reductase catalytic subunit from unknown origin
OaAEPlb: asparginyl endopeptidase from Oldenlandia affinis
OGp: 4-guanidinophenyl
PAL: peptide asparaginyl ligase
PARP: poly(ADP-ribose) polymerase enzyme
PD: Parkinson’s disease
PDK1: pyruvate dehydrogenase kinase 1
pelB: pectate lyase B
Pfa: phosphonodifluoromethylenealanine
PG: protecting group
PH: pleckstrin homology
Pma: phosphonomethylenealanine
POI: protein of interest
PRC2: polycomb repressive complex 2, H3K27 methyltransferase
PrP: prion protein
PSD: post synaptic density
PSD-95: postsynaptic density protein 95
PTEN: phosphatase and tensin homolog
PTM: post-translational modification
PTS: protein trans-splicing
Rma DnaB: intein in the DnaB helicase of Rhodothermus marinus
RNase A: ribonuclease A
SAGA: Spt-Ada-Gcn5 acetyltransferase
SaSrtA: Sortase A from S. aureus
Sce VMA: intein in the vacuolar ATPase subunit of Saccharomyces cerevisiae
SEA: bis(2-sulfanylethyl)amino
SEPL: streamlined expressed protein ligation
SFTI: sunflower trypsin inhibitor-1
SH2: Src Homology 2 domain
SH3: Src Homology 3 domain
SHP-2: Src homology region 2-containing protein tyrosine phosphatase 2
Smt3: SUMO family protein
SPPS: solid-phase peptide synthesis
SpSrtA: sortase A from S. pyogenes
Ssp DnaB: intein in the DnaB helicase of cyanobacterium Synechocystis sp., strain PCC 6803
Ssp DnaE: naturally split intein in the DnaE protein from Synechocystis sp., strain PCC 6803
Ssp DnaX: intein in the DNA polymerase III subunit tau from Synechocystis sp., strain PCC 6803
Ssp GyrB: intein in DNA gyrase subunit B from Synechocystis sp., strain PCC 6803
STL: serine/threonine ligation
SUMO: small ubiquitin-like modifier
SWI-SNF: switch/sucrose nonfermentable, a nucleosome remodeling complex
TBR-I/II: type I/II TGF-P receptor subunits
Ter DnaE-3: naturally split intein in the DnaE protein from Trichodesmium erythraeum
TEV: tobacco etch virus
TFA: trifluoroacetic acid
TGF-β: transforming growth factor-β
TGM2: tissue transglutaminase 2
tRNA: transfer RNA
Trt: triphenylmethyl
Trx: thioredoxin
Ulp1: ubiquitin-like-specific protease
VidaL T4Lh-1 and UvsX-2: atypically split inteins discovered from metagenomic analysis from Lake Vida Brine Hole 2
WWP2: WW domain containing protein 2, an E3 ubiquitin ligase

Biographies

Biographical Sketch

Tom W. Muir received his B.Sc in Chemistry from the University of Edinburgh in 1989 and his Ph.D. in Chemistry from the same institute in 1993 under the direction of Professor Robert Ramage. Following postdoc studies with Stephen B.H. Kent at The Scripps Research Institute, Muir joined the faculty of The Rockefeller University in 1996, where he rose through the ranks eventually being appointed the Richard E. Salomon Family Professor and Director of the Pels Center of Chemistry, Biochemistry and Structural Biology. In 2011, Dr. Muir joined the faculty of Princeton University as the Van Zandt Williams Jr. Class of ‘65 Professor of Chemistry. He currently serves as Chair of the Chemistry Department. Dr. Muir has published many articles in the areas of peptide and protein chemistry. His current interests lie principally in the area of epigenetics, where he is interested in how changes to chromatin structure drive different cellular phenotypes.

Robert E. Thompson received his received his Ph.D. in Chemistry from the University of Sydney in 2014. During his graduate studies under the supervision of Professor Richard J. Payne and Professor Katrina A. Jolliffe, Rob developed chemical methods for the synthesis of post-translationally modified bioactive peptides. In 2015, he joined the laboratory of Professor Tom W. Muir at Princeton University where he is developing and applying tools in chemistry and protein engineering to study chromatin.

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PERMALINK

Chemoenzymatic Semisynthesis of Proteins

Robert E Thompson

Tom W Muir

Abstract

1. INTRODUCTION

Figure 1.

Figure 2.

2. CHEMICAL LIGATION OF EXPRESSED PROTEINS

2.1. Native Chemical Ligation

Figure 3.

2.1.1. Chemical synthesis of N-terminal cysteinyl peptides

Figure 4.

2.1.2. Chemical synthesis of peptide α-thioesters

Figure 5.

2.1.3. Expression of N-terminal cysteinyl proteins.

Figure 6.

Figure 7.

Figure 8.

2.1.4. Chemical routes to recombinant protein α-thioesters.

2.2. Inteins and Expressed Protein Ligation

Figure 9.

2.2.1. Mechanism of intein-mediated protein splicing

Figure 10.

2.2.2. Production of recombinant protein α-thioesters using inteins.

Figure 11.

Table 1.

2.3. Extensions and Alternatives to Native Chemical Ligation

Figure 12.

3. PROTEIN TRANS-SPLICING

Figure 13.

3.1. Artificially split inteins

Figure 14.

Figure 15.

Table 2.

Figure 16.

3.2. Naturally split inteins

Figure 17.

3.3. Engineering of split inteins

Figure 18.

4. TRANSPEPTIDATION

4.1. Sortase A

Figure 19.

Figure 20.

Figure 21.

Figure 22.

Figure 23.

4.2. Peptide Asparaginyl Ligases

4.2.1. Butelase 1

Figure 24.

Figure 25.

4.2.2. OaAEP1b

5. PEPTIDE LIGASES

5.1. Subtiligase

Figure 26.

Figure 27.

5.2. Trypsiligase

Figure 28.

6. HYBRID CHEMOENZYMATIC LIGATION TECHNOLOGIES

6.1. Transpeptidase-mediated synthesis of protein thioesters

Figure 29.

6.2. Streamlined Expressed Protein Ligation

Figure 30.

6.3. Enzyme-Catalyzed Expressed Protein Ligation

Figure 31.

6.4. Transpeptidase-Assisted Intein Ligation

Figure 32.

7. APPLICATIONS OF PROTEIN SEMISYNTHESIS

7.1. Chromatin modifications

Figure 33.

7.1.1. Histones

Figure 34.

Figure 35.

Figure 36.

Figure 37.

7.1.2. Asymmetrically Modified Nucleosomes

Figure 38.

Figure 39.

7.1.3. Chromatin-Associated Proteins

7.1.4. Semisynthetic Modification of Native Chromatin