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. 2024 Mar 27;33(4):e4973. doi: 10.1002/pro.4973

Design of a symmetry‐broken tetrahedral protein cage by a method of internal steric occlusion

Nika Gladkov 1, Elena A Scott 2, Kyle Meador 1, Eric J Lee 1, Arthur D Laganowsky 2, Todd O Yeates 1,3,4,✉, Roger Castells‐Graells 4,✉
PMCID: PMC10966355  PMID: 38533546

Abstract

Methods in protein design have made it possible to create large and complex, self‐assembling protein cages with diverse applications. These have largely been based on highly symmetric forms exemplified by the Platonic solids. Prospective applications of protein cages would be expanded by strategies for breaking the designed symmetry, for example, so that only one or a few (instead of many) copies of an exterior domain or motif might be displayed on their surfaces. Here we demonstrate a straightforward design approach for creating symmetry‐broken protein cages able to display singular copies of outward‐facing domains. We modify the subunit of an otherwise symmetric protein cage through fusion to a small inward‐facing domain, only one copy of which can be accommodated in the cage interior. Using biochemical methods and native mass spectrometry, we show that co‐expression of the original subunit and the modified subunit, which is further fused to an outward‐facing anti‐GFP DARPin domain, leads to self‐assembly of a protein cage presenting just one copy of the DARPin protein on its exterior. This strategy of designed occlusion provides a facile route for creating new types of protein cages with unique properties.

Keywords: DARPin, nanoparticles, protein cages, protein design, symmetry‐breaking

1. INTRODUCTION

Advances in protein design have made it possible, with sufficient experimental trials, to generate self‐assembling protein nanoparticles or protein cages, which often take the form of Platonic solids (tetrahedra, cubes, icosahedra) (Aupič et al., 2022; Bale et al., 2016; Cristie‐David et al., 2019; de Haas et al., 2023; Edwardson et al., 2022; Fletcher et al., 2013; Golub et al., 2020; King et al., 2012; King et al., 2014; Lai et al., 2012; Lai et al., 2014; Malay et al., 2019; Meador et al., 2023; Padilla et al., 2001; Sasaki et al., 2017; Sciore et al., 2016; Terasaka et al., 2018). Principles of symmetry have played a critical role in successful strategies for designing protein cages and similar assemblies (Laniado & Yeates, 2020; Sciore et al., 2016; Subramanian et al., 2021; Yeates, 2017). Building symmetric structures minimizes the engineering requirements for achieving robust assembly. That principle was first emphasized by Crick and Watson (Crick & Watson, 1956) in the context of natural assemblies (viral capsids) and was a guiding principle in developing the first method for designing novel protein cage assemblies (Padilla et al., 2001).

A consequence of design methods that exploit symmetry is that the resulting architectures are repetitive. For example, the outer surface of a symmetric protein cage will present many structurally and chemically equivalent motifs (e.g., many equivalent chain termini) for attachment or fusion—12 for symmetry T, 24 for symmetry O, and 60 for symmetry I. That feature is beneficial for some applications (e.g., vaccine‐like particles (Antanasijevic et al., 2020; Brouwer et al., 2021; Marcandalli et al., 2019; Walls et al., 2020), polyvalent binding (Divine et al., 2021; Miller et al., 2023), enzymatic materials (McConnell et al., 2020; McNeale et al., 2023), and imaging scaffolds (Castells‐Graells et al., 2023; Liu et al., 2018; Liu et al., 2019)). However, for other applications it may be desirable to functionalize (or present fusions) on a singular location. It is notable that this type of “addressability” is generally easy to achieve for nanomaterials based on nucleic acids, where symmetry is typically not a fundamental feature. One particular form of designed protein architectures—protein origami (Ljubetič et al., 2017)—also lacks symmetry. Recent efforts have also succeeded in generating an asymmetric holey viral capsid through a set of feature‐specific hierarchical assembly steps of subunits with designed asymmetry (Zhao et al., 2021). Furthermore, it has been possible to change the symmetry of protein cages by point mutations that alter their subunit‐subunit interactions (Sharma et al., 2022; Szyszka et al., 2024). Nonetheless, self‐assembly design methods, whose successes are dramatically expanding due to modern machine learning algorithms (de Haas et al., 2023; Meador et al., 2023), generally give rise to symmetric architectures, and thus to challenges in breaking symmetry for certain purposes.

One recent study succeeded in breaking the symmetry of a protein cage in order to produce a designed capsid larger than would otherwise be possible with purely icosahedral symmetry (Lee et al., 2023). That effort relied on an exceptionally complex and demanding task of designing a series of similar but selectively associating protein–protein interfaces. Fourteen sets of cage designs were required to identify successful cases of cages with broken symmetry. Motivated by the utility of protein cages with broken symmetry and by the difficulty associated with their creation, we sought a straightforward strategy that might allow the production of protein cages with broken symmetry and singular motifs for exterior binding or attachment.

In the present work, we demonstrate a new way to produce designed protein cages with broken symmetry. Our approach exploits a principle of steric collision in the protein cage interior to assure that only one copy of a modified subunit (bearing an extra inward‐facing domain) can be accommodated in an otherwise symmetric cage. The asymmetry of the resulting cage is validated through biophysical and structural methods, including native mass spectrometry.

2. RESULTS

2.1. Protein design of a symmetry‐broken cage

In designing a tetrahedral‐type protein cage with broken symmetry, we took the previously characterized protein cage known as T33‐51 as a starting point (Cannon et al., 2020). T33‐51 is a tetrahedrally symmetric architecture built from two subunit types, A and B, with stoichiometry A12B12. Four trimers of each subunit type sit on the body diagonals of a cube. The two naturally trimeric components of T33‐51 spontaneously self‐assemble into a supramolecular structure due to a binding association at a designed interface between them.

To develop a new approach for creating symmetry‐broken cages, we introduced a modified protein component A, referred to as A', which includes fusion to a small inward‐facing protein domain. Owing to its inward‐facing fusion, and the limited space in the interior of the protein cage, only one copy of the A' subunit can be incorporated in an assembled cage without steric conflict; the other 11 copies must be occupied by the unmodified subunit A.

A key motivation for breaking the symmetry of protein cages is to enable the display of unique attachments (not subject to symmetric repetition) on the exterior surface. Accordingly, in our design application, we further modified the A' subunit with an outward‐facing fusion to a DARPin protein domain. DARPin domains have been exploited as general binders, as their loop regions can be mutated (on the basis of library selection experiments) to bind diverse target proteins (Boersma & Plückthun, 2011; Hansen et al., 2017; Plückthun, 2015). Our fusion was to an anti‐GFP DARPin, which has been the subject of recent protein cage applications (Castells‐Graells et al., 2023; Liu et al., 2019). This design scheme is shown in Figure 1. To favor the desired assembly result, we co‐transformed Escherichia coli with a gene for protein A' in a low copy vector, with the A and B subunits of the T33‐51 cage expressed on a high copy vector. We expected this scheme to generate a tetrahedral cage that displays a single anti‐GFP DARPin on its surface (Figure 1a,b).

FIGURE 1.

FIGURE 1

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(a) Design of a symmetry‐broken T33‐51 protein cage. Subunits A (green) and B (blue) of the T33‐51 cage were cloned into a high copy vector and co‐transformed along with the A' subunit (pink), which contains an internal SUMO fusion (red) and an outward‐facing anti‐GFP DARPin fusion (violet), in a low copy vector into Escherichia coli. Due to its inward‐facing SUMO fusion, and the limited space in the interior of the protein cage, only one copy of the A' subunit can be incorporated in an assembled cage without steric conflict. (b) Structure of the symmetry‐broken T33‐51 cage. One A trimer contains a symmetry‐broken A' subunit (pink), which displays one anti‐GFP DARPin (purple) on the cage surface and occupies 65% of the internal cavity volume of the cage with the inward‐facing SUMO fusion (red). (c) Structure of symmetry breaking A'. In addition to an N‐terminal (inward‐facing) SUMO domain, the A' subunit is fused to an outward‐facing anti‐GFP DARPin (not shown).

To evaluate inward‐facing domains for fusing to subunit A', we employed AlphaFold2 (Jumper et al., 2021) as a convenient approach for obtaining plausible structural models. As candidates, we curated a set of common protein fusion tags, ranging from 10 to 20 kDa. We reasoned that this size range would allow for the fusion protein to fill a substantial fraction of the internal cavity of the cage without compromising its structural integrity. After reviewing these structure predictions, we selected the SUMO tag to serve as the internal fusion candidate based on its molecular weight, globular tertiary structure, and potential utility for subsequent characterizations. We evaluated models of the SUMO fusions with different linker lengths by aligning each version of the A' subunit to the published crystal structure of the T33‐51 cage (Cannon et al., 2020) in PyMOL, noting whether the internal fusion protein fit within the cage interior without clashing with other A or B components. Based on this modeling, we chose GGSSG as a short flexible linker sequence (Figure 1c). The predicted volume of the SUMO tag is 23,300 Å3. To validate that one SUMO fusion occludes the cage cavity, we calculated the volume of the internal lumen of the cage by approximating it as a sphere with a radius of 20.6 Å. Therefore, the cage cavity has a volume of 37,100 Å3, which we also estimated using CASTp (Tian et al., 2018) (Figure S1). These calculations show that the SUMO fusion occupies approximately 65% of the internal cage cavity volume, allowing for only one A' subunit to fit into the cage (Figure 1b).

2.2. Biochemical characterization of a symmetry‐broken cage

We co‐transformed the original A and B subunit genes for T33‐51 in the high copy pRSFDuet‐1 vector and the A' gene in medium‐to‐low copy pET22b + vector into E. coli BL21(DE3) cells. We purified the cages from cell lysate using immobilized nickel affinity chromatography based on a polyhistidine tail on the B subunit. SDS‐PAGE analysis (Figure S2) confirmed the presence of the symmetry‐broking A' subunit in the presumptive protein cage complex obtained by nickel affinity purification.

Size exclusion chromatography (SEC) of the material obtained from nickel affinity purification confirmed successful cage assembly at the expected elution volume (Figure 2a), falling between that for the unmodified T33‐51 cage and a T33‐51 cage displaying 12 DARPins (one on each A component in the previous symmetric construction) and eluting before the peak corresponding to lower‐order A‐B assemblies. When analyzed with blue‐native PAGE, the fractions eluting at 13 and 14 mL, on the left shoulder of the SEC peak, produced a distinct band at the expected molecular weight of a symmetry‐broken cage (Figure 2b). SDS‐PAGE analysis of the size exclusion chromatography fraction indicated the presence of symmetry‐breaking component A' in the cage peak fractions (Figure 2c), further confirming that the cage peak reflects symmetry‐broken cages. Gel densitometry analysis of the bands corresponding to A, A', and B subunit components confirmed the expected stoichiometric ratio between the A' and A components (Figure 2d). In the selected SEC peak, the experimentally estimated ratio of A' to A was 1.1:10.9, which is in close agreement with the theoretical value of 1:11, showing that only one copy of the A' subunit was incorporated into the overall cage assembly, as expected. In order to verify that only this portion of the SEC peak contained cages, we performed negative‐stain electron microscopy (EM) screens on samples from the 11, 13, and 16 mL SEC elution fractions (Figure S3). Negative‐stain EM of the 11 mL fraction showed predominantly higher‐order assemblies lacking tetrahedral symmetry and aggregates. Cage presence in this fraction was minimal and can be attributed to leakage or imperfect sample separation. Negative‐stain EM of the 13 mL fraction revealed primarily tetrahedrally symmetric assemblies with the expected architectural features of cages, supporting our identification of this fraction as harboring most of the cages. Negative‐stain images of the 16 mL elution fraction showed significantly fewer cages than the 13 mL sample amidst a large quantity of lower‐order assemblies and aggregates (which are likely composed of cage degradation products). These results confirm that symmetry‐broken cages elute primarily in the 13 and 14 mL SEC fractions, as our gel densitometry results taken alone would suggest.

FIGURE 2.

FIGURE 2

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Purification of symmetry‐broken T33‐51 cage. (a) Size exclusion chromatography of immobilized nickel affinity chromatography‐purified symmetry‐broken cage (purple), unmodified T33‐51 cage (teal), and T33‐51 cage with surface anti‐MBP DARPin fusions on all 12 A components (pink). Peaks corresponding to the cage appear from 12 to 14 mL. (b) Blue native PAGE of symmetry‐broken cage size exclusion chromatography elution fractions (elution volume 11–17 mL). (c) SDS‐PAGE of symmetry‐broken cage size exclusion chromatography elution fractions (elution volume 11–17 mL). (d) Gel densitometry of each lane in (c). Molecular‐weight normalized band density was multiplied by 12 to visualize more easily the stoichiometric ratio of T33‐51 components to symmetry‐broken A' in each elution fraction.

To demonstrate that the symmetry‐broken cage does indeed bind GFP with the intended 1:1 (GFP:cage) stoichiometry, we assayed the binding interaction between GFP and the designed cage (Figure 3). SEC of pure symmetry‐broken cage incubated with excess GFP revealed co‐elution of cage and GFP as evidenced by the presence of an absorbance peak (Abs 488 nm) at the elution volume expected for the cage‐GFP complex and distinct from the absorbance peak (Abs 488 nm) at the elution volume expected for monomeric GFP (Figure 3b). Indeed, the SDS‐PAGE analysis of the cage‐GFP complex elution peak confirmed the presence of bands at the molecular weights expected for GFP and cage subunits (Figure 3c). The stability of the obtained cage‐GFP complex is indicated by our observation that after doing a second round of SEC of the purified cage‐GFP complex, the 280/488 nm absorbance ratio for the cage‐GFP elution volume remained nearly the same (Figure 3d). Quantification using a calibration curve showed the number of GFP molecules bound to the symmetry‐broken cage was only slightly diminished. We observed 1.4 equivalents of GFP per cage after the first round of SEC, and 1.1 equivalents after a second round of SEC (Figure 3e). These results show that the obtained symmetry‐broken cage displays a single anti‐GFP DARPin on its surface.

FIGURE 3.

FIGURE 3

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GFP binding assay of the symmetry‐broken cage. (a) Overview of our GFP binding assay approach. Following purification of our symmetry‐broken cage, we incubated the obtained cage sample with excess GFP and isolated the obtained cage‐GFP complex through size exclusion chromatography (SEC). To confirm the stability of this complex, we resubjected the obtained pure cage‐GFP complex to SEC. We measured GFP fluorescence emission at 507 nm in both samples following excitation at 488 nm and compared this emission to a calibration curve of purified cage mixed with known amounts of GFP to calculate the number of GFP molecules bound to each symmetry‐broken cage. (b) 280 nm absorbance and 488 nm absorbance (GFP) traces of the SEC peaks for the cage‐GFP complex and the monomeric GFP. (c) SDS‐PAGE analysis of the cage‐GFP complex and free GFP SEC peaks shown in (b). (d) 280 and 488 nm absorbance traces for the cage‐GFP complex obtained in (b) after a second SEC round of purification. (e) Fluorescence emission GFP binding assay. We generated a calibration curve by mixing pure cage with known amounts of GFP, ranging from 0.1 to 12 GFP molecules for 1 cage, and measuring 507 nm fluorescence emission following 488 nm excitation. We generated a linear trendline from this calibration curve and used this trendline to calculate the number of GFP molecules bound to cage in the samples from the SEC purification steps.

2.3. Structural characterization of the symmetry‐broken cage

We pursued structural studies using EM and native mass spectrometry. Through negative‐strain microscopy (Figure 4a), we confirmed that the designed assembly forms geometrically regular particles of the expected size, reflective of intact cages. We also performed preliminary cryo‐EM analysis. Here as well, images confirmed that intact cages were formed (Figure 4b). A low‐resolution 3D reconstruction resulted in density closely matching the regular T33‐51 cage. Not unexpectedly, image processing (even in the absence of symmetry constraints) did not reveal the singular protruding DARPin domain. Owing to its flexible attachment, small size, and 1/12 occupancy, particle alignment algorithms were not able to uniquely define the singular attachment. That finding provides an interesting counterpoint to recent cryo‐EM scaffolding developments where symmetric and rigid attachments have proven critical for imaging (Castells‐Graells et al., 2023). Similarly, we did not resolve clear density for the encapsulated SUMO domain, though it is possible that higher resolution cryo‐EM structural studies might reveal additional features.

FIGURE 4.

FIGURE 4

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Structural characterization of the symmetry‐broken cage. (a) Negative‐stain electron microscopy of the symmetry‐broken cage. Three particles of the expected size of a T33‐51 cage that exhibit roughly cubic shape are highlighted. The scale bar is 50 nm. (b) Cryo‐electron microscopy of the symmetry‐broken cage. Twelve 2D particle classes are shown, which were used for the low‐resolution 3D reconstruction (left) of the symmetry‐broken cage core.

We turned to native mass spectrometry as a precise method for resolving the subunit stoichiometry of the assembled cages. Native mass spectrometry has proven useful in other studies on designed protein cages (Lai et al., 2014; Sahasrabuddhe et al., 2018; Sasaki et al., 2017; Tamara et al., 2022; Wargacki et al., 2021). Native mass spectrometry further confirmed the architecture and correct stoichiometry of the symmetry‐broken cage (Figure 5). The dominant mass form (observed at ~486 kDa) was within 0.2% of the calculated mass of a symmetry‐broken cage with stoichiometry A'1A11B12 (485 kDa). We note that, without the inward‐facing fusion, the incorporation of A versus A' subunits into a given cage would be largely random and, thus, the number of A' subunits in the cage stoichiometry would resemble a binomial distribution. In contrast, the mass spectrometry result shows the A'1A11 form as the overwhelmingly dominant species. Interestingly, the experimentally observed mass was slightly higher than the calculated model value. We hypothesize that this could reflect the incidental encapsulation of molecular species in the cell (where assembly occurs), and it is notable that a similar deviation was observed in prior work on hollow protein cages (Lai et al., 2014). Future work could endeavor to either eliminate the encapsulated species through a sequential cage disassembly‐rinsing‐reassembly process, or adapt this encapsulation capability to incorporate molecules of interest into the cage cavity.

FIGURE 5.

FIGURE 5

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Native mass spectrometry of symmetry‐broken cage. (a) The architecture of the symmetry‐broken cage. The cage is composed of 11 copies of subunit A (17.1 kDa), 12 copies of subunit B (21.0 kDa), and one copy of subunit A' (44.4 kDa). The total mass of the symmetry‐broken cage is 485 kDa. (b) Native mass spectrum of symmetry‐broken cage in 200 mM ammonium acetate. (c) Deconvolution of mass spectrum shown in (b).

3. DISCUSSIONS AND CONCLUSION

The central aim of this work was to demonstrate a method for generating symmetry‐broken protein cages. Our approach, exploiting a strategy of steric occlusion, was successful in that goal. A modified version of an otherwise tetrahedrally symmetric cage was shown to comprise a single copy of a unique subunit variant, thereby allowing the presentation of a single copy of an outward‐facing binding domain. The relative simplicity of the present design approach is notable. A suitable protein cage having a subunit with an inward‐facing terminus is required, and an appropriate choice must be made for the interior occluding domain, but no other challenging design elements (e.g., novel protein–protein interfaces) were required to achieve the desired goal.

As designed protein cages find increasing uses in nanotechnology and medicine, the ability to prepare asymmetric or addressable variants will become more important. Symmetric versions of protein cages have proven critically useful in single‐particle cryo‐EM applications as imaging scaffolds (Castells‐Graells et al., 2023; Liu et al., 2018; Liu et al., 2019), but asymmetric forms could be advantageous in other imaging applications, including in in‐situ cryo‐EM tomography, where it might be desirable to avoid the effects of high‐copy number binding in a native cellular environment. Accordingly, symmetry‐broken cages with singular binding motifs on their exterior could prove valuable as mid‐nanometer, targeted geometric markers inside cells. Potential targets for this technology are key mitochondrial membrane proteins, such as mitofusin‐1 (MFN1), which have eluded high‐resolution intracellular structural characterization. Structures obtained from this endeavor would provide key insights into diseases and the fundamental biological processes that underlie morphological alterations in mitochondria or other subcellular components (Gee et al., 2024). Those applications are currently under investigation.

4. METHODS

4.1. Design of symmetry‐breaking component A'

A set of common fusion tags was curated and evaluated for suitable size (<20 kDa) and possible utility in the context of cage characterization to serve as internal fusions for the symmetry‐breaking component A'. From this set, the small ubiquitin‐like modifier (SUMO) protein was selected. Next, a set of linker sequences, which included the short linker AQ, the flexible linker GGSSG, the rigid linker EEEAQKAA, and several alpha‐helical linkers between A and the internal fusion were evaluated for the SUMO tag in order to determine which linker would afford the most favorable placement of the internal fusion relative to A and the cage cavity. This evaluation was performed by obtaining structure predictions of the A‐linker‐internal fusion protein using AlphaFold2 (Jumper et al., 2021) and aligning this prediction to the published crystal structure of the T33‐51 cage (PDB ID: 5cy5) in PyMOL (Cannon et al., 2020). The final chosen design for the symmetry‐breaking component A' used the linker GGSSG.

4.2. Cloning and protein expression

A DNA fragment containing the sequence for T33‐51 components A and B separated by a spacer (sequence shown below) sourced from Cannon et al. (2020) was synthesized (Twist Biosciences) and cloned into the pRSFDuet‐1 vector (gifted by Dr. Mark Arbing, UCLA‐DOE IGP) using Gibson assembly cloning. Separately, DNA fragments containing the four chosen symmetry‐breaking A designs were synthesized (Twist Biosciences) and cloned into the pET22b + vector via Gibson assembly. Plasmid DNA for all constructs was cloned in E. coli DH5alpha cells (New England Biolabs) and extracted using ZymoPURE Plasmid Miniprep Kits.

Purified T33‐51 and symmetry‐breaking A constructs were co‐transformed into E. coli BL21(DE3) cells and plated on Luria Bertani broth (LB) agar supplemented with 50 μg/mL kanamycin and 50 μg/mL ampicillin. Three colonies from each plate were used to inoculate 50 mL of LB supplemented with 50 μg/mL kanamycin and 50 μg/mL ampicillin. The LB growths were then incubated with shaking overnight at 37°C and 200 rpm. Following overnight incubation, 10 mL of each growth was used to inoculate 1 L of kanamycin and ampicillin‐supplemented LB. Growths were incubated with shaking at 37°C and 180 rpm until they reached an OD600 of 0.6, at which point expression was induced with 100 μM isopropyl β‐d‐1‐thiogalactopyranoside. Growths were incubated with shaking at 18°C and 180 rpm for 18 h, after which cells were harvested by centrifugation at 4000 × g for 20 min at 4°C.

4.3. Protein purification

Harvested cell pellets were resuspended in lysis buffer (250 mM NaCl, 50 mM Tris–HCl pH 8.0, 20 mM imidazole pH 8.0, 2% w/v glycerol) supplemented with lysozyme, EDTA‐free protease inhibitor cocktail, DNAse, and RNAse (Thermo Fisher Scientific). Cells were then lysed using an Avestin EmulsiFlex C3 homogenizer. Cell lysates were clarified for 30 min at 20,000 × g at 4°C. The clarified cell lysates were loaded onto a column containing equilibrated immobilized nickel affinity chromatography resin (Thermo Fisher Scientific) and eluted using a linear imidazole gradient from 50 to 250 mM. All immobilized nickel chromatography fractions, along with the cell pellet, were analyzed using SDS‐PAGE.

100 mM imidazole and 250 mM imidazole elution fractions were concentrated using Millipore Amicon Ultra 100 kDa molecular weight cutoff filters at 3500 × g and 4°C. Concentrated protein samples were further purified via size‐exclusion chromatography using a Superose 6 Increase column (GE Biosciences) and eluted with SEC buffer (250 mM NaCl, 50 mM Tris–HCl pH 8.0, 2% w/v glycerol). Size exclusion chromatography fractions corresponding to suspected cage peaks were analyzed via SDS‐PAGE and native (non‐denaturing) PAGE.

4.3.1. SEC GFP binding assay

400 μM of purified symmetry‐broken cage was mixed with six equivalents of GFP, incubated for 1 h at 4°C, centrifuged for 15 min at 10,000 × g to remove aggregates, and injected onto a Superose 6 Increase column (GE Biosciences) and eluted with SEC buffer (250 mM NaCl, 50 mM TRIS–HCl pH 8.0, 2% w/v glycerol) in order to separate cage‐GFP complexes from free GFP. SEC fractions corresponding to suspected cage peaks were analyzed via SDS‐PAGE. Purified cage‐GFP complexes were subsequently concentrated and resubjected to size exclusion chromatography using a Superose 6 Increase column.

4.3.2. Fluorescence GFP binding assay

In order to generate the GFP calibration curve, 200 nM of purified symmetry‐broken cage aliquots were mixed with 0.1, 0.2, 0.5, 1, 3, 6, or 12 equivalents of GFP. These samples were incubated for 1 h at 4°C and, subsequently, centrifuged for 15 min at 10,000 × g to remove aggregates. A SpectraMax M5 plate reader was used to excite the GFP in these samples, along with the previously obtained cage‐GFP complex samples (normalized to 200 nM), with 488 nm light, and measured the fluorescence emission at 507 nm. Fluorescence emission data of cage samples with known amounts of GFP was plotted against equivalents of GFP. A linear regression trendline was generated from these data and applied to the fluorescence emission of cage‐GFP complex samples in order to calculate the number of equivalents of GFP bound to each cage in these samples.

4.4. Negative stain electron microscopy

Following size exclusion chromatography, symmetry‐broken cage samples were diluted to 50 μg/μL. Formvar/Carbon 400 mesh copper grids (Ted Pella Inc) were glow‐discharged for 30 s at 15 mA using a PELCO EasiGlow. 5 μL of cage sample was transferred to the glow‐discharged grids. After 30 s of incubation, the sample was removed by blotting, and the grid was stained with 2% uranyl acetate for 30 s. The grid was then imaged using a Tecnai T12 electron microscope.

4.5. Cryo‐electron microscopy

Following size exclusion chromatography, symmetry‐broken cage samples were concentrated and mixed with purified GFP in a final ratio of 1:12 cage to GFP at 0.6 mg/mL final cage concentration. 3.5 μL of the sample mixture was applied to glow discharged Quantifoil 300 mesh R2/2 copper grids and frozen with liquid ethane using a Vitrobot Mark IV (FEI). Grids were imaged using a Talos F200C. Automatic particle picking, 2D classification, and 3D reconstruction were performed using CryoSPARC (Punjani et al., 2017).

4.6. Native PAGE

Following size exclusion chromatography, fractions spanning the obtained cage peak were sampled and diluted 4:1 with ThermoFisher NativePAGE Sample Buffer (4×). The native PAGE was run at 150 V for 105 min at 4°C in ThermoFisher NativePAGE Running Buffer.

4.7. Native mass spectrometry

Size exclusion chromatography fractions corresponding to the symmetry‐broken cage peak were pooled and concentrated to 50 μM. Samples were buffer‐exchanged using Micro Bio‐Spin 6 desalting column (Bio‐Rad) into 200 mM ammonium acetate (pH adjusted to 7.4 with ammonium hydroxide). Samples were diluted to 2 μM and loaded into pulled borosilicate glass capillaries prepared in‐house. Samples were electrosprayed with the voltage applied using a platinum wire directly inserted into the solution into a Q Exactive Ultra‐High Mass Range Hybrid Quadrupole‐Orbitrap mass spectrometer (Thermo Scientific). Instrument resolution was set to 3125, in‐source collision energy dissociation at 30 V, collision energy at 50 V, source temperature at 170°C, capillary voltage at 1.3 kV, source DC offset at 21 V, inter flatapole lens at 10 V, injection flatapole DC at 15 V, bent flatapole DC at 15 V, transfer multiple DC at 1 V, and trapping gas pressure at 7.5.

4.8. Figure visualization

Symmetry‐broken cage structures were created using published crystal structures of T33‐51 A and B components (PDB ID: 5cy5) (Cannon et al., 2020) and AlphaFold2 predictions for the symmetry‐breaking A with the SUMO tag and short linker. These structures were rendered in PyMOL (Version 2.0 Schrödinger, LLC). Gel densitometry analyses were carried out using the Gel Analyzer feature in ImageJ (Schneider et al., 2012).

AUTHOR CONTRIBUTIONS

Roger Castells‐Graells: Methodology; investigation; writing – review and editing; visualization; supervision; formal analysis; software; writing – original draft. Nika Gladkov: Methodology; conceptualization; investigation; formal analysis; visualization; writing – original draft; writing – review and editing; software. Elena A. Scott: Investigation; methodology; formal analysis. Kyle Meador: Conceptualization. Eric J. Lee: Methodology; writing – review and editing. Arthur D. Laganowsky: Supervision; resources; funding acquisition. Todd O. Yeates: Conceptualization; writing – review and editing; writing – original draft; funding acquisition; supervision; resources; project administration.

FUNDING INFORMATION

This work was supported by the US Department of Energy Office of Science award DE‐FC02‐02ER63421. This work was also supported by Welch Foundation (A‐2106‐20220331) and NIH (R01GM139876 and RM1GM1454316) awarded to A.L.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

Supporting information

Data S1. Supporting Information.

PRO-33-e4973-s001.docx (1.9MB, docx)

ACKNOWLEDGMENTS

The authors thank Mark Arbing for providing the pRSFDuet‐1 vector and the GFP, for granting access to a SpectraMax M5 plate reader, and for helpful discussions regarding cloning. We thank Duilio Cascio and Alex Lisker for computing support. We thank Soichi Wakatsuki for helpful discussions on the subject of symmetry‐breaking.

Gladkov N, Scott EA, Meador K, Lee EJ, Laganowsky AD, Yeates TO, et al. Design of a symmetry‐broken tetrahedral protein cage by a method of internal steric occlusion. Protein Science. 2024;33(4):e4973. 10.1002/pro.4973

Reviewing Editor: Aitziber L. Cortajarena

Contributor Information

Todd O. Yeates, Email: yeates@mbi.ucla.edu.

Roger Castells‐Graells, Email: rcastellsg@ucla.edu.

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Supplementary Materials

Data S1. Supporting Information.

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