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. 2005 Jan;14(1):64–73. doi: 10.1110/ps.04965405

Orthogonal site-specific protein modification by engineering reversible thiol protection mechanisms

J Jefferson Smith 1, David W Conrad 1, Matthew J Cuneo 1, Homme W Hellinga 1
PMCID: PMC2253321  PMID: 15576565

Abstract

Covalent modification is an important strategy for introducing new functions into proteins. As engineered proteins become more sophisticated, it is often desirable to introduce multiple, modifications involving several different functionalities in a site-specific manner. Such orthogonal labeling schemes require independent labeling of differentially reactive nucleophilic amino acid side chains. We have developed two protein-mediated protection schemes that permit independent labeling of multiple thiols. These schemes exploit metal coordination or disulfide bond formation to reversibly protect cysteines in a Cys2His2 zinc finger domain. We constructed a variety of N- and C-terminal fusions of these domains with maltose-binding protein, which were labeled with two or three different fluorophores. Multiple modifications were made by reacting an unprotected cysteine in MBP first, deprotecting the zinc finger, and then reacting the zinc finger cysteines. The fusion proteins were orthogonally labeled with two different fluorophores, which exhibited intramolecular fluorescene resonance energy transfer (FRET). These conjugates showed up to a threefold ratiometric change in emission intensities in response to maltose binding. We also demonstrated that the metal- and redox-mediated protection methods can be combined to produce triple independent modifications, and prepared a protein labeled with three different fluorophores that exhibited a FRET relay. Finally, labeled glucose-binding protein was covalently patterned on glass slides using thiol-mediated immobilization chemistries. Together, these experiments demonstrated that reversible thiol protection schemes provide a rapid, straightforward method for producing multiple, site-specific modifications.

Keywords: covalent modification, biosensor, fluorescence resonance energy transfer, surface immobilization

Covalent modification is an important natural (Han and Martinage 1992; Kukuruzinska and Lennon 1998; Johnson 2004) and biotechnological (DeSantis and Jones 1999; Qi et al. 2001) strategy to introduce new functionalities into proteins. Examples include cofactors for catalysis (Kaiser 1988; Tann et al. 2001), the use of fluorophores (Marvin et al. 1997) or electrochemical (Benson et al. 2001) groups for detection of ligand binding in biosensors, and immobilization on solid surfaces (Domen et al. 1990; Willner et al. 2002). It is frequently necessary to modify the protein site-specifically to optimally combine the conjugated functionality with the intrinsic properties of the protein. As demands on the functionalities of engineered proteins become more sophisticated, it is often desirable to introduce multiple, covalent modifications involving several different functionalities in a site-specific manner. Strategies to produce proteins with single or multiple nonnatural amino acids include total synthesis (Jantz and Berg 2003), semisynthesis by ligation of synthetic and biologically expressed fragments (Muir et al. 1998; Hofmann et al. 2001; Hofmann and Muir 2002), and in vitro translation using a partially extended genetic code (Zhang et al. 2003, 2004). Nevertheless, one of the simplest methods still remains covalent modification of biologically expressed proteins (Hermanson 1996). This strategy requires a single, uniquely reactive amino acid. Cysteine is well suited for this purpose, since it is relatively rare, and the thiol(ate) presents a uniquely reactive functional group that is readily modified under mild conditions (Hermanson 1996). Multiple, independent site-specific modifications require more than one differentially reactive cysteine. In rare cases these occur in naturally evolved proteins, permitting different labels to be introduced independently (Tanaka et al. 1997). Engineered cysteine pairs have also been used (Ha et al. 1999; Ratner et al. 2002; Schuler et al. 2002; Rhoades et al. 2003; Allen et al. 2004), but typically have insufficient differential reactivity to obtain highly specific double labeling and require additional purification steps to separate the various labeled contaminants. Here we present a scheme to engineer proteins with multiple, differentially reactive cysteines that are independently addressable through reversible thiol protection (RTP) mechanisms.

Cysteines that are oxidized in a disulfide bridge, or that coordinate to a metal, are often protected from covalent modification by thiol-reactive reagents. Protection is readily reversed by reduction or metal chelation. It is therefore possible to engineer proteins with multiple, independently addressable, site-specific covalent attachment points by constructing several cysteines that are (1) unprotected, (2) react to form a disulfide, and (3) participate in metal binding. We have developed a strategy that allows two or three sites to be independently modified by fusing a protein with a single, unprotected cysteine with one or two small domains that contain a Zn2+-binding site or a disulfide bridge (Fig. 1 ▶). These fusion domains are based on a consensus zinc-finger domain, ZifQNK (Shi and Berg 1995). This 32-residue domain has a Cys2His2 primary coordination sphere that binds Zn2+ reversibly with 10−9–10−11 M affinity (Michael et al. 1992). In the absence of Zn2+, the two cysteines can form a disulfide under oxidizing conditions (Knapp and Klann 2000). ZifQNK can therefore be used in either metal-dependent or redox-dependent RTP strategies (MRTP, RRTP). Additionally, we used a truncated, 18-residue version of ZifQNK (βZIF) in which the single α-helix bearing the two histidines has been deleted, leaving a two-stranded β-sheet containing the two cysteines that readily oxidize to form a disulfide, but do not bind Zn2+ in the reduced form. Thus, βZIF can be used in a RRTP strategy.

Figure 1.

Figure 1.

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Schemes for producing multiple, site-specific modifications in zinc finger fusion proteins using either reversible metal coordination or disulfide-mediated protection strategies. Two distinct thiol reactive modifications are represented as ⋆ and ▴.

To demonstrate these different schemes we constructed fusions with Escherichia coli maltose-binding protein (MBP) that has a single cysteine engineered at position 141, and glucose-binding protein (GBP) that has a single cysteine engineered at position 149. MBP and GBP are members of the periplasmic binding protein (PBP) superfamily (Tam and Saier Jr. 1993; de Lorimier et al. 2002). They are soluble, monomeric receptors that consists of two domains linked by a hinge region (Quiocho and Ledvina 1996). The proteins adopt at least two conformations—an open, ligand-free state, and a closed, ligand-bound state—that interconvert upon ligand binding via a hinge-bending motion. MBP, GBP, and other members of the PBP superfamily have been used to construct reagentless fluorescent and electrochemical sensors by covalently coupling single fluorescent (de Lorimier et al. 2002) or redoxactive (Benson et al. 2001) reporter groups, respectively, that respond to the ligand-mediated conformational changes. These motions can also be coupled to changes in fluorescence resonance energy transfer (FRET) between fusions of suitable derivatives of green fluorescent protein (GFP) at the N and C termini of MBP (Fehr et al. 2002) and other PBPs (Fehr et al. 2004). Here we construct fusions of ZifQNK or βZIF at the N or C termini of MBP, and demonstrate that these can be used to obtain ligand-responsive FRET between donor and acceptor fluorophores site specifically coupled at position 141 within MBP (MBP141) and the fusion domain. We also construct a FRET relay (Watrob et al. 2003) between three fluorophores in a triply labeled, double-fusion protein.

The immobilization of proteins on glass, gold, or other nonbiological substrates is an important aspect of constructing hybrid devices such as biosensors (Willner and Katz 2000, 2003; Willner et al. 2002); it is also an increasingly important component for the construction of protein chips used in genome analysis technologies (Figeys and Pinto 2001). Orientation-specific immobilization using defined attachment points on a protein has numerous advantages over random, multipoint chemi- or physisorption (Lu et al. 1996; Rao et al. 1998; Turkova 1999), especially in cases where binding sites need to be presented, or conformational changes are taken advantage of, such as is the case for the proteins presented in this work. Again, site-specific thiol-mediated covalent linkage strategies offer advantages over noncovalent site-specific linkages such as provided by a oligohistidine C- or N-terminal fusions (Gershon and Khilko 1995; Allard et al. 2002). Here we demonstrate that GBP first labeled with a fluorophore at the unprotected cysteine 149 can be patterned on a glass slide by covalent coupling using reversibly protected cysteines in a ZifQNK fusion peptide.

Results

Independent double labeling can be achieved using amino-or carboxy-terminal fusions of either ZifQNK or βZIF to protein with a single, unprotected cysteine (Fig. 1 ▶). In the case of ZifQNK either MRTP, or RRTP strategies can be used; for βZIF, only RRTP is possible. Independent triple labeling can be achieved using a fusion with both ZifQNK (MRTP) and ZifQNK (RRTP).

Differential reactivity of engineered thiols

The multiple labeling scheme requires that protected thiols are significantly less reactive than unprotected thiols, and that protection is reversible. To test this, we reacted cysteine-free MBP (MBPwt), MBP141, MBPwt fused at the C terminus with ZifQNK in the Zn2+ form (MBPwt::ZifQNK•Zn), in the Zn2+-free oxidized form (MBPwt::ZifQNKox), and in the Zn2+-free reduced form (MBPwt::ZifQNKred) with 7-diethylamino-3-(4′maleimidylphenyl)-4-methyloumarin (CPM). CPM becomes fluorescent upon covalent conjugation (Parvari et al. 1983). The reactions were carried out in parallel under typical conditions used for labeling proteins, measuring the increase in fluorescence upon formation of the conjugate (Table 1). Cysteine-free MBPwt shows very slight reactivity, presumably due to reaction with surface lysines, since maleimides react slowly with primary amines as well as thiols (Hermanson 1996). The metal- and oxidatively-protected thiols in MBPwt::ZifQNK•Zn and MBPwt::ZifQNKox react with CPM at the same very slow rate as detected for the thiol-free protein. The unprotected thiols in MBP141, and MBPwt::ZifQNKred react 10,000-fold more rapidly than the protected thiols, with the reaction being >95% complete in 10 min or 30 min, respectively. Both metal- and redox-mediated strategies therefore provide excellent protection and are readily reversible.

Table 1.

Reaction rates for the conjugation of 7-diethylamino-3-(4′maleimidylphenyl)-4-methyloumarin (CPM) to protected and deprotected cysteines

Protein t1/2 (min)
MBPwt 31,100
MBP141C 2.5
MBPwt::ZifQNK•Zn 27,500
MBPwt::ZifQNKox 28,800
MBPwt::ZifQNKred 5.8
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Double labeling

To investigate site-specific labeling with two different fluorophores, C-terminal ZifQNK fusions with MBP141 were constructed with a thrombin-cleavable peptide linker (MBP141::tb::ZifQNK). Cy5 maleimide mono-reactive dye and tetramethylrhodamine-5-maleimide (TMR) were used as the fluorescent labels. Both the metal- and redox-mediated protection strategies were used to generate the two possible labeling combinations (i.e., a total of four experiments): first attachment of Cy5 to the unprotected Cys141, followed by deprotection (chelation or reduction) and attachment of two TMR labels to ZifQNK (MBP141(Cy5)::tb::ZifQNK(TMR)2; and addition of label in the reverse order to generate MBP141(TMR)::tb::ZifQNK(Cy5)2.

After the first reaction, the protein:fluorophore ratio was determined by absorbance spectroscopy, and was found to be approximately 1:1 in all four cases, consistent with complete reaction of the unprotected thiol in MBP141, and full protection of the two thiols in the ZifQNKox or ZifQNK•Zn2+ domain. In the second reaction, the ZifQNK was first deprotected by addition of chelator or reductant, and reacted with the other fluorophore. The stoichiometry of the reaction was determined by absorbance spectroscopy and mass spectrometry (Fig. 2 ▶; Table 2). In all four cases, the ratios were 1:1:2 for protein:fluorophore 1:fluorophore 2, consistent with the expected labeling pattern. The masses were also as expected for the appropriately labeled protein (Table 2). We also separated the labeled MBP141 and ZifQNK domains by thrombin cleavage of the linker peptide to determine the degree of mislabeling (first fluorophore on ZifQNK; second fluorophore on MBP141) by the optical absorbance and retention times of the fragments (Fig. 2 ▶). In all four cases, no evidence of mislabeling was observed. Taken together, these results are therefore consistent with the intended, site-specific, double-labeling patterns, and show that both redox- and metal-mediated reversible thiol protection strategies work well with ZifQNK.

Figure 2.

Figure 2.

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Analysis of labeling patterns in MBP141C(Cy5)::th::ZifQNK(TMR)2 and MBP141C(TMR)::th::ZifQNK(Cy5)2 as indicated. (A) Absorbance spectra of doubly labeled proteins. Spectra of the conjugate produced by metal-mediated protection shown at half the concentration of those produced by the disulfide-mediated scheme. Calculated ratios for MBP141C(Cy5)::th::ZifQNK(TMR)2 with disulfide protection are Cy5/protein = 1.09 and TMR/Cy5 = 2.05 and with metal protection are Cy5/protein = 1.07 and TMR/Cy5 = 2.18. Ratios for MBP141C(TMR)::th::ZifQNK(Cy5)2 with disulfide protection are TMR/protein = 0.97 and TMR/Cy5 = 0.57 and with metal protection are TMR/protein = 0.98 and TMR/Cy5 = 0.52. (B) HPLC chromatographs of thrombin cleaved MBP141C(Cy5)::th::ZifQNK(TMR)2 and MBP141C(TMR)::th::ZifQNK(Cy5)2 produced by the disulfide-mediated scheme. Metal-mediated multiple-labeling scheme have identical chromatographs (not shown). The three chromatographs represent the same HPLC run monitored at different wavelengths: 280 nm for peptide, 525 nm for TMR, and 650 nm for Cy5. The triple peaks that elute around 10 min are the Zif peptides, and the single peak at 23 min is the MBP peptide. (C) Mass spectra of the doubly labeled MBP141::th::ZifQNK proteins.

Table 2.

Masses of modified proteins and peptide fragments

Polypeptide Theoretical mass (Da)a Experimental mass (Da)b
MBPwt::ZifQNK 46,213 46,200
MBP141(TMR)::th::ZifQNK(Cy5)2 48,250 48,317
MBP141(TMR)c 41,820 41,873
ZifQNK(Cy5)c 5657 5663
ZifQNK(Cy5)2c 6435 6446
MBP141(Cy5)::th::ZifQNK(TMR)2 47,953 47,814
MBP141(Cy5)c 42,117 42,157
ZifQNK(TMR)c 5360 5386
ZifQNK(TMR)2c 5842 5866
βZif(IAF)2::th::MBP141(TMR)::th::ZifQNK(Cy5)2 51,417 51,578
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a Theoretical masses calculated using DNA Strider version 1.2.

b Experimental masses measured using MALDI-TOF mass spectrometer, as described in Materials and Methods.

c Peptide fragments obtained by thrombin cleavage.

FRET in doubly labeled proteins

Both types of doubly labeled protein exhibited a maltose-dependent decrease in FRET between the TMR donor and Cy5 acceptor fluorophores (Fig. 3 ▶). The distances between the attached fluorophores is expected to be less in the ligand-bound closed conformation than in the open conformation of the apo-protein. It is therefore likely that orientation, rather than distant-dependent effects, dominate the FRET mechanism in this system (Lakowicz 1999). Furthermore, the magnitude of the change differs in the two constructs: MBP141(TMR)::tb::ZifQNK(Cy5)2 shows a threefold change in the ratio of the donor:acceptor emission intensities upon addition of maltose, whereas MBP141(Cy5)::tb::ZifQNK(TMR)2 shows only a 0.1-fold change. The maltose affinities of the labeled and unlabeled proteins are similar (Fig. 3 ▶), indicating that the two fluorophores did not significantly perturb the conversion between the open and closed conformations.

Figure 3.

Figure 3.

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Intramolecular FRET between TMR and Cy5 of MBP141C(Cy5)::th::ZifQNK(TMR)2 and MBP141C(TMR)::th::ZifQNK(Cy5)2. (A) Emission spectra obtained in the presence (dashed line) and absence (solid line) of maltose (excitation at 540 nm). Spectra at intermediate maltose concentrations are shown for MBP141C(TMR)::th::ZifQNK(Cy5)2. Note the presence of an isosbestic point. (B) Titration curves of maltose binding reported as change in the ratio of the summed emission intensities of the donor (560–640 nm) and acceptor (642–700 nm) fluorophores. The measured Kd values are 0.2 μM and 2 μM, respectively.

Triple labeling

To investigate labeling with three different fluorophores, we constructed a MBP141 with βZif fused to the N terminus, and ZifQNK to the C terminus, using a thrombin-cleavable linker peptide in each case (βZif::tb::MBP141::tb::ZifQNK). βZif and ZifQNK form an orthogonally protected pair: Redox-mediated protection has to be used for βZif, mandating the metal-mediated strategy for ZifQNK in this case. The order in which modifications and deprotections are carried out is important: (1) The unreacted thiol is modified; (2) βZifox is deprotected by reduction, and modified; (3) ZifQNK•Zn2+ is deprotected by chelation, and modified. Steps 2 and 3 cannot be inverted, because deprotection of ZifQNK•Zn2+ requires addition of reductant, which would also deprotect βZifox.

Cy5, TMR, and 5-iodoacetamide fluoroscein (IAF) were used as the labels. Two proteins with different labeling patterns were prepared using the appropriate order of modification and deprotection steps: βZif(IAF)2::tb::MPB141(Cy5)::tb::ZifQNK(TMR)2 and βZif(IAF)2::tb::MPB141(TMR)::tb::ZifQNK(Cy5)2. Labeling stoichiometries were determined by absorbance spectroscopy for the single and double modifications, but not for the triply labeled proteins, due to the spectral overlap of TMR and IAF (Fig. 4A ▶). The stoichiometry was also confirmed by measuring the mass of triple modified protein (Table 2). We determined the degree of mislabeling by cleaving both N- and C-terminal fusions with thrombin and separating the labeled products on HPLC (data not shown). The unprotected cysteine and the ZifQNK cysteines were exclusively modified with the correct fluorophores. The βZif cysteines were correctly labeled with at least one IAF. The IAF reaction did not quite reach completion (~90%), however, leaving the second cysteine in some of the βZif fusions free to react with the fluorophore in the third modification.

Figure 4.

Figure 4.

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Preparation and analysis of triply labeled MBP conjugate. (A) Absorbance spectra of double-labeled intermediate, βZif(IAF)2::th::MBP141C(Cy5)::th::ZifQNK, (dashed line) [Cy5/protein ratio = 1.06 and IAF/Cy5 ratio = 1.82] and triple-labeled final product, βZif(IAF)2::th::MBP141C(Cy5)::th::ZifQNK(TMR)2. (B) Emission intensity spectrum demonstrating the FRET relay effect (exciting IAF at 490 nm). Emission from IAF is observed at 525 nm, TMR at 580 nm, and Cy5 at 670 nm. The apo form is indicated by a solid line and the maltose saturated form is indicated by a dashed line.

FRET in triply labeled proteins

IAF/TMR and TMR/Cy5 both constitute FRET pairs. It is therefore possible to construct an intramolecular FRET relay where excitation energy can be transferred from IAF to Cy5 via TMR (Fig. 5 ▶). As predicted, βZif(IAF)2::tb::MPB141(Cy5)::tb::ZifQNK(TMR)2 demonstrated a complete FRET relay but βZif(IAF)2::tb::MPB141(TMR):: tb::ZifQNK(Cy5)2 did not, presumably because the separation between IAF and TMR is within the Förster distance in βZif(IAF)2::tb::MPB141(Cy5)::tb::ZifQNK(TMR)2 (42 Å) but exceeds the Förster distance in βZif(IAF)2::tb::MPB141(TMR)::tb::ZifQNK(Cy5)2 (61 Å). FRET between TMR and Cy5 still occurs in βZif(IAF)2::tb::MPB141(TMR)::tb::ZifQNK(Cy5)2 when TMR is excited (50 Å). The FRET relay demonstrated a maltose-dependent decrease (Fig. 4B ▶).

Figure 5.

Figure 5.

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Confocal microscopy images of GBP149C(Cy5)::ZifQNK covalently patterned on BMOE modified glass slides (A) and GBP149C(Cy5) nonspecifically absorbed on BMOE modified glass slides (B). Light gray corresponds to Cy5 fluorescence and indicates surface-bound protein. The grid bars are where BMOE was protected from photooxidation by the copper mask. The square pits are areas that were photooxidized.

Protein immobilization

GBP149::ZifQNKox was derivatized with Cy5 at Cys149. The disulfide was reduced and GBP149(Cy5)::ZifQNKred was reacted with a glass slide patterned with bis-maleimidoethane (BMOE) (Fig. 5A ▶). The BMOE pattern was generated by protecting thiol silane from photooxidation with a 10-μm beehive mask as described in Materials and Methods. An image of a slide prepared with a Cy5-modified GBP lacking the ZifQNK fusion was also taken (Fig. 5B ▶). As can be seen, the GBP149(Cy5)::ZifQNK gave the expected square grid pattern corresponding to reaction with the maleimide, whereas the pattern produced by the control protein was significantly dimmer, and is consistent with physisorption of the protein in the irradiated squares where there is a preponderance of negatively charged groups resulting from photooxidation (Bhatia et al. 1992).

Conclusions

We have demonstrated that fusions with one or two zinc finger derivatives allow two or three sites to be modified independently by reversible thiol protection schemes that exploit metal coordination or disulfide formation. We demonstrated that both methods produce orthogonal protein modifications with no apparent mislabeling. Both MBP141(TMR)::th::ZifQNK(Cy5)2 and MBP141(Cy5)::th:: ZifQNK(TMR)2 were rapidly produced by simply switching the order of reactants, unlike many competing methods which require additional synthesis steps (Hofmann and Muir 2002; Zhang et al. 2003).

Both labeling combinations resulted in ligand-induced FRET decreases. MBP141(TMR)::th::ZifQNK(Cy5)2, in particular, generated a larger ligand-mediated signal change than any previously reported intramolecular FRET biosensor (Hofmann et al. 2001; Fehr et al. 2002, 2003; Lager et al. 2003). The large FRET change cannot be explained in terms of distance-dependent effects because the distance change is too small and because the separation between fluorophores gets smaller upon ligand binding, which should produce an increase rather than a decrease in FRET. Instead, we propose that the observed FRET change is due to an orientation effect (Lakowicz 1999). The 2:1 ratio of fluorophores did not appear to interfere with FRET or correct immobilization. We have also demonstrated that both protection methods can be combined to triple modify proteins, and in this case, produce an intramolecular protein FRET relay. FRET relays have utility in overcoming large distances (Watrob et al. 2003) and provide large Stokes shifts. Another use for the triple modification strategy may be to immobilize a FRET biosensor to produce a ratiometric device. We have demonstrated that different modifications can be combined to immobilize Cy5 modified proteins in an orientation-specific pattern.

ZifQNK and βZif fusions are a rapid, straightforward way to add functionalities to almost any protein. The protection strategies are not limited to these domains, however. Other suitable domains containing disulfides or stable metal centers can be used. Furthermore, the metal-mediated protection scheme could be extended to any thiol protected by a tightly binding ligand. Finally, the approach can be even further generalized by using design methods to introduce disulfides (Ivens et al. 2002; Nemeth et al. 2002), metal centers (Hellinga 1998), or ligand binding sites (Looger et al. 2003) in suitable locations.

Materials and methods

Clone construction

The peptide sequences used for ZifQNK C-terminal and βZif N-terminal fusions with the thrombin cleavage sites were: GLVPR|GSTGEKPYKCPECGKSFSRSDHLSRHQRTHQNKKG SHHHHHH and MTGEKPYKCPECGKSFSRSLVPR|GSGG, respectively (cysteines indicated in bold; linker peptide underlined; thrombin recognition site italicized; cleavage site indicated with |). The C-terminal zinc finger fusion was generated by PCR using the following oligonucleotides:

5′-GGAGGTTCAACAGGTGAGAAACCGTACAAGTGCC CGGAGTGTGGCAAATCATTCTCTCGATCGGACCAT, 5′-CG GGATCCTATCACTTCTTGTTCTGATGTGTCCGTTGGTGACG GGATAGATGGTCCGATCGAGAGAATG, and 5′-CTCACCTG TTGAACCTCCCTTGGTCAGCTTAGTCTG. The N-terminal βZif was constructed by PCR using the following oligonucleotides: 5′-GGAATTCCATATGACAGGTGAGAAACCGTACAAGTG CCCGGAGTGTGGC and 5′-CCTTCTTCGATTTTGCCCCCGG ATCCTCGAGGGACGAGCGATCGAGAGAATGATTTGCCA CACTCCGGGCA. Wild-type MBP was used as a template to generate the zinc-finger fusions. The MBP A141C mutant was generated by PCR using the following oligonucleotides: 5′-GAAC TGAATGCAAAGGTAAGAGCGCG and 5′-CGCGCTCTTACC TTTGCATTTCAGTTC. All recombinant constructs were cloned into pET21a for expression.

Protein expression and purification

Recombinant proteins were overexpressed in BL21(DE3). One liter of 2xYT was inoculated with 25 mL from a culture freshly grown to stationary phase (9 h), and grown at 37°C to an optical density of A600 = 0.4, induced with 1 mM IPTG, and grown for a further 2 h. The cultures were supplemented with 100 μM ZnCl2 at induction to ensure viability. For MBP fusions, cell pellets were resuspended in IMAC buffer (20 mM MOPS, 500 mM NaCl, 10 mM imidazole [pH 7.5]), lysed by sonication (2 min), and a cleared lysate produced by centrifugation (25 min, 25,000g). The MBP fusions were purified using nickel-charged IMAC resin followed by gel filtration (Superdex 200). Pure protein was quantified by absorbance (ɛ280 = 66,000 M−1cm−1).

Labeling reaction kinetics

Proteins (1 μM in 50 mM MOPS, 100 mM NaCl [pH 6.0]) were reacted with a fivefold molar excess of CPM (concentrated stock solution in DMSO). The labeling reaction was monitored by following the increase in fluorescence at 470 nm (excitation 385 nm) for the CPM-protein conjugate as a function of time using a fluorescence plate reader (SprectraMAX GeminiXS, Molecular Devices). The values for t1/2 were obtained from fits of the data using a commercial software package (TableCurve 2D, SYSTAT Software, Inc.). All experiments were conducted at 25°C.

Metal-mediated reversible thiol protection

Proteins were exchanged from purification buffer into modification buffer (50 mM MOPS, 100 mM NaCl [pH 6.0]) by gel filtration (Superdex 200). For the first modification (unprotected thiol), 25 μM protein was incubated (room temperature, 30 min; agitated with a roller drum) with 125 μM TCEP, 100 μM ZnCl2, and 250 μM tetramethylrhodamine 5-maleimide or Cy5 dye in a total volume of 1 mL. The reaction then was transferred to a desalting column (BioRad PD10) preequilibrated with modification buffer, collecting the first colored band (modified protein). The labeling efficiency of the first modification was determined as described below. The second pair of thiols were deprotected by chelation in the presence of 5 mM EDTA and 2 mM orthophenathroline (4°C; 8 h). Following removal of the chelators by gel filtration (Superdex 200), the protein was labeled with 500 μM TMR or Cy5 dye in the presence of 250 μM TCEP, (1-mL reaction volume; 1 h, room temperature; agitated on a roller drum). Unincorporated label was removed by a desalting column (BioRad PD10), eluting with 50 mM MOPS, 100 mM NaCl (pH 6.8).

Redox-mediated reversible thiol protection

To chelate any free metal, purified protein was first incubated with 5 mM EDTA and 2 mM o-phenanthroline (4°C, 8 h), followed by exchange into 20 mM Tris, 100 mM NaCl (pH 6.0) on a S200 gel filtration column. In these preparations, the disulfide in the ZifQNK peptide was completely oxidized, as determined by DTMB reactivity. For the first modification (unprotected thiol), 25 μM protein was incubated with 250 μM TMR or Cy5 dye (1-mL reaction volume; room temperature for 30 min; agitated on a roller drum). Free fluorophore was removed by desalting column (see above), and the labeling efficiency was determined as described below. Deprotection by reduction and dye modification were carried out in one step by the addition of 250 μM TCEP and 500 μM Cy5 or TMR (1 h at room temperature). Unreacted material was removed by desalting column (see above).

Triple modification

The unprotected thiol was labeled first using 25 μM protein and 250 μM Cy5 (30 min at room temperature; agitated on a roller drum). After removing unreacted fluorophore by gel filtration (see above), the βZif domain was deprotected and labeled (125 μM TCEP and 250 μM 5-IAF; 30 min at room temperature). Excess 5-IAF was removed by gel filtration. The ZifQNK domain was deprotected by chelation with 5 mM EDTA and 2 mM o-phenanthroline (8 h at 4°C), followed by gel filtration and labeling protein with 150 μM TCEP and 250 μM TMR. The triple labeled product was purified from excess fluorophore by gel filtration (see above).

Determination of fluorophore labeling stoichiometry

Dye–protein ratios were determined using:

graphic file with name M1.gif

where Afluor. is the absorbance at 650 nm for Cy5 and 525 nm for TMR, Aprotein is the absorbance at 280 nm, ɛprotein = 66,000 M−1cm−1, ɛfluor. is 250,000 M−1cm−1 for Cy5, 95,000 M−1cm−1 for TMR and 75,000 M−1cm−1 for 5-IAF, and N is 0.05 (Amersham Biosciences) for Cy5 and 0.3 for TMR.

The equation for dye/dye ratios was:

graphic file with name M2.gif

where Afluor1 is the absorbance for fluorophore 1, Afluor2 is the absorbance for fluorophore 2, ɛfluor1 is the extinction coefficient for fluorophore 1, and ɛfluor2 is the extinction coefficient for fluorophore 2.

Thrombin cleavage and HPLC purification

Protein was cleaved with biotinylated thrombin according to the manufacturer’s protocol (Novagen Thrombin Cleavage Capture Kit). The cleavage products were separated by HPLC (Waters 2795 Alliance HT, PDA detector) using a C4 reversed phase column (Symmetry 300), eluting with a linear gradient from 20% B to 100% B over 80 min at a flow rate of 1 mL/min (A = water with 0.1% TFA; B = acetonitrile with 0.1% TFA). Peaks were identified by absorbance and elution times. Assignments were confirmed by MALDI-TOF mass spectrometry (Applied Biosystems, Voyager DE).

Fluorescence spectroscopy

Fluorescence emission intensities were measured at 25°C in a stirred 1-cm quartz cell using a fluorimeter (AMINCO Bowman Series 2). Protein samples were diluted to 0.2 μM using 20 mM MOPS, 100 mM NaCl (pH 7.0 buffer). Excitation for TMR and IAF was 530 nm and 490 nm, respectively. Fluorescence emission spectra were collected from 550 nm to 700 nm.

Protein immobilization and confocal imaging

A glass slide was silanized with a 20:1 ratio of bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane:3-mercaptopropyltrimethoxysilane. A pattern was then produced by photooxidation of the 3-mercapto-propyltrimethoxysilane with short wavelength ultraviolet irradiation for 5 min in the presence of a copper mask (10-μm square beehive). Thiols that were protected from photooxidation by the mask were reacted with a homobifuctional crosslinker, bis-maleimidoethane (BMOE). The cysteines in ZifQNK were then de-protected with TCEP, and the GBP149(Cy5)::ZifQNK incubated with the slide to react with the maleimide of BMOE. After 1 h, the substrate was washed with buffer to remove uncoupled protein, and imaged using a Zeiss LSM-410 confocal microscope.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04965405.

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