Site-Specific Cross-Linking of Proteins through Tyrosine

Bioconjugate Chem. 2005, 16, 1617−1623

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Site-Specific Cross-Linking of Proteins through Tyrosine Hexahistidine Tags R. Scott Stayner, Dong-Joon Min,‡ Patrick F. Kiser, and Russell J. Stewart* Department of Bioengineering, 20 South 2030 East, Room 506, University of Utah, Salt Lake City, Utah 84112 . Received August 12, 2005

The genetic addition of hexahistidine (H6) tags is widely used to isolate recombinant proteins by immobilized metal-affinity chromatography (IMAC). Addition of a tyrosine residue to H6 tags enabled proteins to be covalently cross-linked under mild conditions in a manner similar to the natural, sitespecific cross-linking of tyrosines into dityrosine. A series of seven hexahistidine tags with tyrosines placed in various positions (H6Y tags) were added to the amino terminus of the I28 immunoglobulin domain of the human cardiac titin. The H6Y-tagged I28 dimerized in the presence of excess Ni2+ with a KD of 200 µM. Treatment of Ni2+-dimerized H6Y-I28 with an oxidant, monoperoxyphthalic acid (MMPP) or sodium sulfite, resulted in covalent protein multimerization through chelated Ni2+-catalyzed cross-linking of the Y residues engineered into the H6 tag. The protein oligomerization was observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). The presence of dityrosine in the cross-linked proteins was confirmed by fluorescence emission at 410 nm. Proteins lacking the Y residue in the H6 tag treated with the same oxidative conditions did not cross-link or exhibit dityrosine fluorescence, despite the presence of an endogenous Y residue. The method may have potential uses in other protein conjugation applications such as protein labeling and interfacial immobilization of proteins on artificial surfaces.

INTRODUCTION

The expanding use of recombinant proteins in several major industries feeds a growing need for new technologies for labeling, cross-linking, and interfacial immobilization of active proteins. Current protein conjugation and cross-linking methods have several major limitations. Most rely on diffusible reagents that react somewhat specifically with a particular class of nucleophilic functional group, such as the thiol of cysteines or primary amine of lysines. One problem with this approach is the lack of selectivity where all accessible groups of a particular class are modified. This can create multiple compounds, and if one of the targeted functional groups is in or near the active site, the target protein can be inactivated. For purposes of interfacial immobilization, this lack of selectivity can lead to random orientations and conjugation inefficiency. A further drawback of nonselectively modifying an entire class of functional groups is that specific protein modification cannot be carried out in a mixture with several other proteins. Therefore, relatively large quantities of purified protein are required for chemical modifications. All of these problems could be overcome with the development of a conjugation chemistry that utilizes nondiffusible, reactive groups at a genetically specified site on the target protein. Looking to nature, one can find many examples of natural materials composed of highly cross-linked proteins and other biopolymers (1-7). The extensive covalent cross-linking gives these materials their impressive tensile strength, elasticity, tear resistance, toughness, * Corresponding author. Phone: (801) 581-8581. Fax: (801) 581-8966. E-mail: [email protected]. ‡ Current address: Division of Pediatric Hematology-Oncology, New York University School of Medicine, New York, NY 10016.

and other critical material properties. A common natural strategy to cross-link biopolymers is the localized oxidation of specific amino acid side chains by metalloenzymes to form reactive intermediates that then spontaneously form cross-links within a restricted space. An example of this strategy is the oxidative deamination by lysyl oxidase of specific lysine residues that then condense with vicinal nucleophiles to covalently cross-link collagen and elastin (8, 9). Another example is oxidative coupling of the phenolic moiety of two tyrosines, which is catalyzed by several structurally and mechanistically distinct categories of metalloenzymes, including peroxidases, tyrosinases, and laccases (10-13). Dityrosine protein linkages have been found in many structural proteins including elastin (1), silk, and plant cell wall extensin (3, 4), and in hardened fertilization membranes of insect and sea urchin eggs (7). Tyrosine residues are not a common target for sitespecific chemical modification of proteins in vitro because they are generally unreactive under physiological pH. However, there have been reports of in vitro dityrosine cross-linking based on metal-catalyzed oxidation of tyrosine phenolic side chains that are reminiscent in some respects to the natural tyrosine cross-linking process described above (14-17). Peptidic complexes of Ni2+ in the presence of peracid oxidizing agents catalyze the formation of tyrosine free radicals that then react with proximal tyrosine residues to form dityrosine or isodityrosine cross-links (9, 14-17). Brown and co-workers (9) found that Ni2+ complexed with the tripeptide GGH catalyzed the cross-linking of dimeric proteins in the presence of the relatively strong oxidant monoperoxyphthalic acid (MMPP) through natural tyrosine residues. Similarly, when GGH was genetically appended to the dimeric protein, ecotin, intermolecular cross-linking occurred between tyrosine residues in the presence of

10.1021/bc050249b CCC: $30.25 © 2005 American Chemical Society Published on Web 11/01/2005

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Stayner et al. EXPERIMENTAL PROCEDURES

Figure 1. A. Schematic diagram of the protein cross-linking strategy. i. Formation of a ternary complex between Ni2+ and two His6-tagged proteins. ii. Addition of an oxidant to trigger H6Ni2+-catalyzed dityrosine cross-linking. iii. Addition of ETDA to remove Ni2+. B. Titin 128 model protein engineered with H6GY N Terminus 1, the proposed structure of the cross-linked protein dimer 2 after oxidation and treatment with ETDA to remove complexed Ni2+.

MMPP and Ni2+ (8). The tyrosine cross-linking efficiency was shown to be dependent on the distance between tyrosines in these experiments. In wild-type ecotin, in which natural tyrosines at the dimer interface are separated by 20 Å, the cross-linking efficiency was 15%. In ecotin genetically modified to move the tyrosines to within 5 Å of one another at the dimer interface, the cross-linking efficiency was 60%. Similarly, the hexapeptide H6, which is routinely used as a tag for purification of recombinant proteins by immobilized metal affinity chromatography (18, 19), has also been shown to be redox active when complexed with Ni2+. The dimer of H6-tagged glutathione S-transferase (H6GST) was covalently cross-linked by dityrosine formation in the presence of Ni2+ and MMPP (14). Proteins that do not naturally associate with H6GST were not crosslinked, demonstrating that H6-mediated cross-linking did not proceed through a highly diffusible reactant but instead was localized to the vicinity of the H6 tag. Going further, the same research group demonstrated that, in the presence of MMPP and Ni2+, H6GST mediated the formation of dityrosine from free tyrosine, that the mutagenic removal of tyrosine residues from H6GST decreased cross-linking efficiency, and that chemical addition of tyrosine-like residues, using the BoltonHunter reagent, increased cross-linking efficiency. In this paper, we take this biomimetic strategy a step further by developing methods for site-specific dityrosine cross-linking of proteins that do not normally associate in solution. Using the I28 immunoglobulin domain from human cardiac titin as a model protein, we show that H6-tagged I28 domains reversibly self-associate in solution in the presence of Ni2+ and that the complexed Ni2+ will catalyze the formation of dityrosine cross-links between Y residues strategically placed in or near the H6 tag (Figure 1).

Preparation of the H6(Y) Titin I28 Plasmids. The construction of the double H6 tagged protein, H6-titin I28-H6 pET24a was previously published (20). The constructs containing the various H6-tyr (H6Y) tags (Table 1) were prepared by amplifying the I28 region with PCR primers, in separate reactions, that incorporated the DNA sequence of the H6Y tag into the 5′ end of the PCR product along with NdeI and XhoI restriction sites for cloning. The PCR products were ligated into pET 26b (Novagen, Inc.) after digestion with NdeI and XhoI and transformed into DH5R. The constructs were verified by DNA sequencing. Expression and Purification of H6YI28 Proteins. For expression, the pET26b-I28 plasmids were transformed into E. coli strain BL21(DE3) (Novagen, Inc.) by electroporation (EPX Genetics, ECM 395). Single colonies from agar plates containing 50 µg/mL kanamycin were grown overnight at 37 °C in 5 mL LB media with 50 µg/ mL kanamycin. LB cultures (500 mL) with 50 µg/mL kanamycin were then inoculated with the overnight cultures and grown at 37 °C until A600 was between 0.6 and 1.0 (about 2 h). Expression of the target protein was induced by adding isopropyl β-D-thiogalactoside (IPTG) to a final concentration of 0.1 mM followed by shaking for 4 h at 37 °C. The cells were then centrifuged (Beckman JA 10 rotor) at 4500 rpm for 15 min at 4 °C. The cell pellets were stored at -70 °C. Frozen pellets were resuspended in 5 mL of binding buffer (50 mM NaH2PO4, 500 mM NaCl, pH 7.5) per gram of pellet. Phenylmethanesulfonyl fluoride (PMSF) and Triton ×100 were added to concentrations of 1 mM and 0.1% v/v, respectively. The cell suspension was sonicated (Fisher 550 Sonic Dismembranator) at power level 4 on ice for 10 s followed by 1 min of cooling, which was repeated six times. The insoluble cell components were pelleted by centrifugation at 20 000 g (Beckman J2-HS Centrifuge, JA-17 rotor) for 45 min at 4 °C. The supernatant was mixed with 2 mL of Ni2+ nitrilotriacetic acid (NTA) agarose beads (Qiagen) equilibrated with binding buffer, allowed to settle in a column and drained by gravity. The collected flow-through was reapplied to the Ni-NTA column. The column was washed with several column bed volumes of the washing buffer (15 mM NaH2PO4, 500 mM NaCl, 60 mM imidazole, pH 7.5). H-tagged proteins were eluted into1 mL aliquots of the elution buffer (15 mM NaH2PO4, 500 mM NaCl, 300 mM imidazole, pH 7.5). The purity of the protein fractions was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) using 15% polyacrylamide gels. Combined fractions were desalted into phosphate-buffered solution (PBS) (50 mM NaH2PO4, 150 mM NaCl, pH 7.5) on PD 10 sepharose columns (Pharmacia). After desalting, the proteins were aliquoted and stored at -70 °C. I28 proteins were concentrated using a 3000 MW cutoff centrifuge filter (Amicon). Protein concentrations were measured using a Coomasie protein assay (Biorad) with bovine serum albumin (BSA) as a standard. Each protein sample was concentrated so that the subsequent crosslinking reactions could be performed in a total volume of 20 µL, with a total protein concentration of 20 µM (4 nmol). Sedimentation Equilibrium. Sedimentation equilibrium experiments were conducted in a Beckman Optima XL-A analytical ultracentrifuge, with an AnTi60 rotor at 20 °C, using six-channel, 12 mm thick, charcoalepon centerpieces. Prior to the analysis, the protein samples were dialyzed against fresh PBS for 20 h. The three sample channels in each cell contained three

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Site-Specific Protein Cross-Linking Table 1. Relative Fluorescence after Ni2+/Sodium Sulfite Treatment protein name

fluorescence, 410 nm (normalized)a

protein sequence

YH6-I28 MNSYH6GMA-titin I28 0 YGH6-I28 MNSYGH6GMA-titin I28 0 YGYGH6-I28 MNSYGYGH6GMA-titin I28 0 H6GYG- I28 MH6GYG-titin I28 0.4 H6GYGYG-I28 MH6GYGYG-titin I28 1 YGH6GY-I28 MNSYGH6GYA-titin I28 0.3 H3GYGH3-I28 MNSH3GYGH3GMA-titin I28 0 H6-I28-H6 MRGSH6GMA-titin I28-H6 0 titin I28 sequence: GSPLIFITPL SDVKVFEKDE AKFECEVSRE KTFRWLKGTQ EITGDDRFEL IKDGTKHSMV IKSAAFEDEA KYMFEAEDKH TSGKLIIEGI LE a The sequences of the H Y tags and the relative emission fluorescence at 410 nm of each of the proteins in the presence of Ni2+ after 6 treatment with the Na2SO3. The complete sequence of I28 is shown in the bottom panel with the endogenous Y residue in bold.

different concentrations (1.4 µM, 4.3 µM, and 13 µM) of either the H6GY-titin I28 protein without added nickel or that had been incubated overnight at 4 °C with 10fold excess nickel(II) acetate. The reference channels contained PBS. Samples were centrifuged until sedimentation and chemical equilibrium were attained. Values of v-bar and the extinction coefficients for each protein were calculated from the amino acid sequence using the method of Laue (21). Various models describing the concentration distribution were fit to final absorbance versus radius data using nonlinear least squares techniques and the analysis program NONLIN (22, 23). NONLIN performs simultaneous nonlinear least-squares fits to one or more sets of ultracentrifuge data. Oxidative Cross-Linking of H6Y-I28 Proteins. All cross-linking reactions were performed in PBS (50 mM NaH2PO4, 150 mM NaCl, pH 7.5). The nickel(II) acetate, monoperoxyphthalic acid (MMPP), and Na2SO3 stock solutions were prepared in DDI H2O. To determine the effect of the position of Y on cross-linking efficiency, the series of H6Y-I28 proteins (Table 1) were mixed in a final volume of 20 µL with nickel(II) acetate, 20 µM, and MMPP at final concentrations of 20, 200, or 200 µM. After addition of nickel(II) acetate, the solution was left on ice for 30 min, then warmed to room temperature before the addition of MMPP to initiate cross-linking. After 2 min, the cross-linking reaction was quenched by addition of 5 µL of 5× SDS-PAGE sample buffer (0. 25 M Tris pH 6.8, 1% SDS, 2% bromophenol blue, 50% glycerol, 10% β-mercaptoethanol). The samples were then analyzed using SDS PAGE. The extent of cross-linking of H6GYI28 as a function of oxidant and oxidant concentration was determined by cross-linking in 4 mM, 400 µM or 40 µM MMPP, or 40 mM, 4 mM, or 400 µM Na2SO3. The reactions were quenched by the addition of 5 µL of 5× sample buffer containing 10% β-mercaptoethanol. The samples were then analyzed by SDS PAGE. Fluorescence Spectroscopy. The formation of dityrosine in the Ni2+/Na2SO3 treated H6GY-I28 proteins was verified using fluorescence spectroscopy. The crosslinking reactions were performed in a 1 mL quartz crystal cuvette at room temperature in a final volume of 400 µL PBS using 15 µM H6GY-I28 and 150 µM of nickel(II) acetate. With an excitation wavelength of 325 nm the fluorescence emission was scanned from 350 to 600 nm with an excitation slit width of 10 nm, emission slit width of 5 nm, and a scan speed of 500 nm/min on a PerkinElmer LS 55 fluorimeter. As a control, fluorescence emission was scanned in the absence of Ni2+. To verify that the fluorescence of cross-linked H6GY-I28 was due to dityrosine, the emission spectrum was compared to the emission spectrum of dityrosine produced by a laccase enzyme purified from pleurotus ostreatus, (Earthfax Development,7324 South Union Park Ave, Midvale, UT

84047, 938 units of activity/mg laccase). Laccase, at a final concentration of 0.3 mg/mL (281 U/mL) was reacted with 1.25 mM L-tyrosine in 600 µL PBS. Dityrosine formation catalyzed by the GGH/Ni2+ complex and Na2SO3 was also analyzed. L-Tyrosine (15 µM) in DDI H2O was combined with a solution of GGH and Ni(II) acetate (150 µM each, DDI H2O) such that the concentration of the GGH tripeptide and the Ni2+ concentration was 10 times that of the L-tyrosine concentration. The solution was allowed to sit at room temperature for 10 min, after which Na2SO3 was added for a final concentration of 1.5 mM. Dityrosine formation was followed using fluorescence spectroscopy. As a control, the preceding experiment was performed in the absence of the GGH tripeptide to demonstrate that Ni2+ and Na2SO3 alone are not sufficient to catalyze the reaction. RESULTS

Self-Association of H6-Tagged I28 Immunoglobulin Domains. It has been reported that a H10-tagged fusion protein oligomerizes in the presence of Ni2+ (24). SDS PAGE analysis showed a ladder of proteins oligomerized into high molecular species that had a stoichiometry of three and six with respect to the monomeric protein. The oligomers were readily converted to the monomeric species by using excess EDTA to remove Ni2+ or by boiling.. Control experiments in which the protein was incubated in the buffer without added Ni2+ did not result in oligomerization. Enzymatic removal of the H10 tags from the fusion protein prevented oligomerization, indicating that oligomer formation involved Ni2+/H10-tag coordination complexes. The titin I28 protein used in these experiments is an Ig domain found in cardiac titin (26). In muscle the physical properties of I28, and other Ig domains, help titin act as an elastic spring that keeps the sarcomere in register (27) by partially unfolding when the muscle is subjected to tensile stress (28). Previous SDS PAGE analysis of I28 have shown that the protein does not selfassociate in vitro (28). To investigate if H6- I28 dimerizes through the H6 tags in the presence of Ni2+, the native molecular weight of I28 was determined by equilibrium sedimentation in the absence and presence of Ni2+. As titin Ig domains are not known to spontaneously associate in vivo (27) or in vitro (29) it was not unexpected that in the absence of Ni2+, an H6-I28 protein behaved as a 12.3 ( 1 kDa monomer (Figure 2A), which is consistent with the sequence derived molecular weight of 11.7 kDa. The H6GY-titin I28 protein in the presence of a 10-fold excess of Ni(II) acetate is weakly associated into a dimer with a dissociation constant of approximately 200 µM (Figure 2B). Oxidative Cross-Linking through H6Y-tags. The known redox activity of the H6Ni2+ complex and the H6-

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Stayner et al.

Figure 2. A. Sedimentation equilibrium of H6GY-titin I28 protein without Ni2+. The lower panels show experimental data points for three different loading concentrations of each protein with the corresponding calculated curve fit (solid line). The H6GY-titin I28 protein is fit to a monomer model, MW ) 12.3 ( 1 kDa. The upper panels show the residuals for these fits, which are small and random indicating a good fit to the monomer model. B. Sedimentation equilibrium data H6GY-titin I28 protein with a 10-fold molar excess of Ni2+. The lower panels show experimental data points for three different loading concentrations of each protein with the corresponding calculated curve fit (solid line). In the presence of Ni2+, the H6GY-titin I28 protein fit a monomer-dimer model with an equilibrium KD of approximately 200 µM. The upper panels show the residuals for these fits, which indicate a good fit to the monomer-dimer model.

Ni2+-mediated self-association of I28 suggested that the ternary Ni2+ complex might catalyze site specific oxidative cross-linking of I28 if Y residues were placed near the H6-tags (Figure 1). To investigate this possibility and to optimize Y placement, a series of H6-I28 proteins were constructed that had Y residues strategically positioned within or adjacent to the H6-tags (Table 1). Solutions of I28 in the presence of Ni2+ were treated with the oxidizing agent MMPP and the efficiency of I28 crosslinking was investigated by SDS-PAGE (Figure 3). The H6Y-tags in which the Y residues were on the inside, or carboxyl end, of the H6 tag (lanes 4 and 5) were covalently cross-linked into dimers predominantly, but also into trimers and higher molecular weight species. These multimers remained intact after vigorous boiling and in the presence of the reducing agent, β-mercaptoethanol, indicating that neither metal-mediated complexes nor disulfide bonds were responsible for the observed interprotein cross-linking. The H6-tags in which the Y residues are on the outside, or amino end, of the H6 tag (lanes 1-3 and 6-7) underwent little cross-linking. In the absence of Ni2+ or MMPP, no cross-linking occurred. A control I28 protein that did not have a Y residue in the H6 tag was not cross-linked in the presence of Ni2+ and MMPP although it does have a single endogenous Y residue (lane 8). These results demonstrate first that H6complexed Ni2+ will catalyze covalent cross-linking between HY-tagged I28, and second that the position of the Y residue relative to the H6-tag has a profound effect on the covalent cross-linking efficiency. Covalent cross-linking of H6GY-I28 protein was also induced by sodium sulfite (Na2SO3) in the presence of Ni2+ (Figure 4). Although the precise mechanism of this process is unknown, XXH peptide/Ni2+ complexes in the presence of SO3- and dioxygen have been shown to

Figure 3. Protein cross-linking efficiency depends on the placement of the Y residue(s) near the H6 tag. Proteins with the Y residue on the inside of the H6 tag, lanes 4 (MH6GY-titin I28) and 5 (MH6GYGY-titin I28) showed efficient cross-linking when treated with MMPP. However, those versions of the protein in which Y residue(s) were placed within or on the amino side of the H6 tag underwent little or no cross-linking when subjected to the same conditions: lanes 1 (YH6-titin I28), 2 (YGH6-titin I28), 3 (MNSYGYGH6-titin I28), 6 (YGH6GY-Titin I28), 7 (H3GYGH3-Titin I28). Lane M: molecular weight markers. Lane 8: no Y control protein (H6-Titin I28-H6).

generate sulfur oxyradicals, e.g. SO3•-, SO5•-, and SO4•(30). The sulfite radical anion SO3•- is known to react rapidly with dioxygen to form the short-lived monoperoxysulfite radical anion SO5•- which can then dimerize to form the more stable sulfate radical anion SO4•- (for a detailed discussion of the metal-catalyzed redox chemistry of Na2SO3 see ref 30). Alternatively, an oxidized nickel metal complex (Ni3+/Ni2+) may also be acting as the phenolic oxidizing agent. One or more of these reactive intermediate species could then oxidize the Y

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Site-Specific Protein Cross-Linking

Figure 4. Effect of oxidant concentration on I28 cross-linking. (A). MMPP-induced cross-linking. Lanes 1-4: H6GY-titin I28. Lanes 5-8: H6-titin I28-H6 no Y control. The final concentration of MMPP was 0 in lanes 1 and 5, 40 µM in lanes 2 and 6, 400 µM in lanes 3 and 7, and 4 mM in lanes 4 and 8. (B) Na2SO3 induced cross-linking. Lanes 1-3: H6GY-titin I28. Lanes 4-6: H6-titin I28-H6 control. The final concentration of Na2SO3 was 400 µM in lanes 1 and 4, 4 mM in lanes 2 and 5 with 40 mM in lanes 3 and 6. Lane M: molecular weight markers.

from L-tyrosine with laccase. In the absence of oxidant or Ni2+ there was little or no fluorescence (Figure 5). These results confirm that the covalent cross-linking in the H6Y-I28 proteins was due, at least in part, to the formation of dityrosine near the H-tags. Furthermore, the relative fluorescence intensity of the oxidatively crosslinked H6Y-I28 proteins correlated with the degree of cross-linking observed by SDS-PAGE; the most crosslinked protein (Figure 3) had the highest fluorescence (Table 1). DISCUSSION

Figure 5. Dityrosine fluorescence. (9) L-Tyrosine and laccase enzyme. (0) H6GYG-titin I28 treated with Ni2+ and Na2SO3, (4) L-tyrosine treated with GGH/Ni2+/Na2SO3, (2) H6GYG-titin I28 treated with Na2SO3 (no Ni2+ control), ([) L-tyrosine treated with Ni2+ and Na2SO3 (no ligand control).

phenol ring residue, resulting in the formation of a phenolic free radical species, which could then undergo oxidative cross-linking with an adjacent phenolic moiety. This scheme is thermodynamically possible as the oneelectron redox potential of [(XXH)Ni2+]-Ni3+/Ni2+ (∼+800 mV) (30), SO3•-/SO32- (+390 mV) (31), SO5•-/SO52- (+840 mV) (31), and SO4•-/SO42- (+2900 mV) (30) are all potent enough oxidizing agents to oxidize phenolic residues (+300 mV) (32). Dityrosine Formation. As further evidence that covalent cross-linking was occurring through the formation of dityrosine bonds, the cross-linked H6Y-I28 proteins were analyzed by fluorescence spectroscopy. Dityrosine has an excitation maximum at 325 nm and a fluorescence maximum at 410 nm (34). Laccase is an enzyme that catalyzes the polymerization of phenol derivatives in nature, such as urushiol found in oriental lacquers (35). Laccase has been used to create dityrosine linkages in Y-containing peptides(36) and to polymerize free phenol, the functional group on tyrosine (11). Reaction of Ltyrosine with laccase to create an internal dityrosine standard resulted in a broad peak of fluorescence at about 400 nm (Figure 5). As an additional control, dityrosine was formed by reacting L-tyrosine with Ni2+ complexed by the tripeptide, GGH, which has been shown previously to generate dityrosine (8, 37). The oxidatively cross-linked H6Y-tagged I28 proteins had fluorescence spectra similar to dityrosine formed

The goal of this work was to develop an alternative method to modify proteins in a site-specific manner under mild conditions. Our approach was predicated on two previously reported observations. First, H-tagged proteins can associate through a ternary complex formed between the H-tags and Ni2+ (24). Second, H6 complexed Ni2+ catalyzes oxidative covalent cross-linking of tyrosine residues into dityrosine (9, 14, 16). We reasoned that it should be possible to use H6-tags to first form a metalmediated complex between H6-tagged proteins and that the metal complex could then be used to catalyze the localized cross-linking of tyrosine residues placed in the vicinity of the metal complex. That H6-tagged proteins associate in solution in the presence of Ni2+ was confirmed by sedimentation equilibrium analysis. H6-tagged I28 existed in a monomerdimer equilibrium in solution with a KD of approximately 200 µM (Figure 2). The Ni2+-mediated association through H6-tags brings genetically placed Y residues into close proximity to one another and the catalytic Ni2+ H6 complex. Addition of an oxidant generates reactive oxidative species, which then generate tyrosine free radicals that react with proximal tyrosines and other residues. By localizing two tyrosine residues in space by the ternary complex, we envisioned the possibility of observing enhanced rates of reaction as the local concentration of tyrosine could be higher than what could be obtained in solution. This rate enhancement affect has been recently observed in reactants tethered to DNA dimers (38, 39). Additionally, the ternary metal complex prevents the reactive free radicals from diffusing away and thereby localizes the cross-linking reaction. The position of the Y residue in the H6-tag had a profound effect on the cross-linking efficiency of the reaction (Figure 3). Placement of the Y residue(s) on the carboxy side of the H6 tag facilitates the cross-linking whereas the placement of Y residue(s) near the amino terminus resulted in little or no cross-linking. Although the mechanism that leads to this difference is not known,

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it may be related to the half-life of the Y free radical generated in the reaction which can vary considerably depending on the local environment of the radical (40, 41). Our data indicates that the His-tagged proteins were dimerized via the formation of Y-Y cross-links, there is also evidence of side reactions. Metal-catalyzed oxidation has been reported to induce peptide bond cleavage in proteins (42-44), sometimes near Y residues (45, 46). Mass spectrometry analysis after trypsin digestion of the H6GY I28 proteins subjected to the oxidative treatment revealed the presence of small peptides conjugated to the H6GY tag digest fragment (data not shown). Peptides containing Y residues generated by the cleavage of the protein near Y residue in the H6 tag likely cross-linked with intact proteins, which accounts for the increased width of the monomer band in the SDS PAGE results (Figure 4). These same results, however, indicate that though the protein cleavage does occur the predominant product is protein cross-linking. It is conceivable that assay conditions such as oxidizing species, oxidant concentration, dioxygen concentration, and the reaction time could be optimized to enhance cross-linking while reducing protein cleavage. The presence of dityrosine was detected by fluorescence spectroscopy in the cross-linked proteins (Figure 5). However, other residues in the H6 tag region are likely to have reacted with the Y free radical. Of the residues vicinal to the Y in the H6 tag, G is a likely candidate since it was shown previously to react with Y in proteins subjected to metal-catalyzed oxidation (37). The strong fluorescence observed indicates that the ladder of high molecular weight proteins observed in the SDS-PAGE studies is mainly due to the formation of Y-Y crosslinking; however, other Y-amino acid cross-links likely contributed to the oligomerization as well. We demonstrated that the H6 tag in conjunction with Ni2+ caused the protein to associate weakly into dimers. However, the association was transient and therefore only observable by sedimentation equilibrium studies. There are a number of residues that can participate in metal binding complexes: E, Y, H, R, K, D, M, C (47). Combinations of these residues in protein tags have the potential to yield stronger metallopeptide complexes than polyhistadine tags. For example, it has been reported that the HNRYGCGCC protein tag was shown to have a stronger metal binding capacity when compared to the common hexahistadine tag and yielded 20% more protein after purification during immobilized metal affinity chromatography (48). The focus of future research will be to develop methods to screen for additional peptides for the propensity to form metal-mediated complexes and catalyze oxidative dityrosine cross-linking triggered by low concentrations of a mild oxidant. In conclusion, the metal-catalyzed oxidative crosslinking system described with further optimization to limit side reactions may circumvent some of the major limitations of conventional protein chemical modification methods. First, the conjugation site is preestablished as a ternary complex before functional groups are created by a mild oxidant. The conjugation site itself catalyzes the creation of the reactive species, which localizes covalent bond formation to the intended region. Therefore, the target functional group is not active in mild reaction conditions, unlike the C residues sometimes used in protein conjugation reactions (49, 50), and the site limits random modifications that may damage or inactivate the target protein. Second, the conjugation site on the protein is genetically encoded in the form of a metal

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chelating peptide. Therefore, it is unnecessary to purify the target protein as a prerequisite for modification; it can be modified within a complex mixture of proteins. The implication for protein array applications is that recombinant proteins may be selectively captured onto a solid support from a crude lysate of cells expressing the protein. Purification and posttranslational chemical modification will be unnecessary. ACKNOWLEDGMENT

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