Control of a Tyrosyl Radical Mediated Protein Cross-Linking Reaction

Jul 20, 2012 - The electrostatic interaction between positive and negative charges clearly shows both attractive and repulsive forces between charges;...
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Control of a Tyrosyl Radical Mediated Protein Cross-Linking Reaction by Electrostatic Interaction Kosuke Minamihata,† Masahiro Goto,†,§ and Noriho Kamiya*,†,§ †

Department of Applied Chemistry, Graduate School of Engineering, and §Center for Future Chemistry,Kyushu University S Supporting Information *

ABSTRACT: Herein, we demonstrate the control of protein heteroconjugation via a tyrosyl coupling reaction by using electrostatic interaction. Aspartic acid and arginine were introduced to a tyrosine containing peptide tag (Y-tag) to provide electrostatic charge. Designed negatively or positively charged Y-tags were tethered to the C-terminus of Escherichia coli alkaline phosphatase (BAP) and streptavidin (SA), and these model proteins were subjected to horseradish peroxidase (HRP) treatment. The negatively charged Y-tags showed low reactivity due to repulsive interactions between the Y-tags with the negatively charged BAP and SA. In contrast, the positively charged Y-tags showed high reactivity, indicating that the electrostatic interaction between Y-tags and proteins significantly affects the tyrosyl radical mediated protein cross-linking. From the heteroconjugation reaction of BAP and SA, the SA with the positively charged Y-tags exhibited favorable cross-linking toward negatively charged BAP, and the BAP-SA conjugates prepared from BAP with GY-tag (GGGGY) and SA with RYR-tag (RRYRR) had the best performance on a biotin-coated microplate. Encompassing the reactive tyrosine residue with arginine residues reduced the reactivity against HRP, enabling the modulation of cross-linking reaction rates with BAP-GY. Thus, by introducing a proper electrostatic interaction to Y-tags, it is possible to kinetically control the heteroconjugation behavior of proteins, thereby maximizing the functions of protein heteroconjugates.

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INTRODUCTION The protein−protein heteroconjugation reaction is one of the principal techniques in biotechnology, enabling the generation of new biomaterials that have distinct functions of the building block proteins.1−4 The heteroconjugation has to be wellcontrolled to maximize the function of the resulting heteroconjugates and also to avoid the loss of function. Therefore, techniques that provide controlled protein−protein heteroconjugation are of great importance, and many heteroconjugation methods have been utilized including the leucine zipper,5 production of fusion proteins, 6 and protein ligation using intein,7,8 while new heteroconjugation methods are still being developed.9−11 One of the most promising ways to achieve a controlled heteroconjugation reaction is the use of enzymatic reactions.12 With the high substrate specificity of enzymes, the cross-linking of proteins can be oriented to a desired position in a target protein. Also, the cross-linking can be carried out under mild conditions, minimizing the loss of protein function. The enzymes that have been extensively used for the purpose of protein heteroconjugation reactions are transglutaminase (TGase)13 and sortase A (SrtA).14,15 There are other candidates that can be used for protein−protein heteroconjugation, such as hAGT,16 PFTase,17 and PPTase;18 yet, these enzymes require introduction of small chemical substrates to target proteins. TGase and SrtA require only short peptidyl substrates, usually less than 20 amino acids; therefore when combined with genetic modification of the target protein, the © 2012 American Chemical Society

use of TGase and SrtA has become a reliable technique to achieve protein heteroconjugation. Following these established enzymatic modification methods, we have reported a protein heteroconjugation reaction using the horseradish peroxidase (HRP) mediated oxidative tyrosinecoupling reaction.4 As described in the previous report, a peptide tag containing tyrosine residues (Y-tag: the sequence of GGGGY, which is abbreviated as GY-tag in this report) was genetically introduced at the C-terminus of Escherichia coli alkaline phosphatase (BAP) and streptavidin (SA) derived from Streptomyces avidinii. Both the GY-tagged BAP and SA became reactive to the enzymatic reaction of HRP, and a high crosslinked BAP-SA heteroconjugate via the Y-tag was obtained. However, since the protein heteroconjugation reaction was based on a homocoupling reaction of tyrosyl radicals, it was difficult to control the conjugation efficiency between BAP and SA. Stewart and co-workers demonstrated the control of a tyrosyl radical homocoupling reaction by using a hexahistidineNi2+ complex, in which a cross-linkage forms between the proximally positioned tyrosine residues of the complex to yield a protein dimer.19 Another possible way to control the tyrosinecoupling reaction could be the use of a leucine zipper,5,20 which has been intensively exploited for artificial protein dimerization. The Fos and Jun families of eukaryotic transcription factors Received: March 18, 2012 Revised: July 5, 2012 Published: July 20, 2012 1600

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form a heterodimer,21 yet they can also homodimerize, which might cause aggregation when the leucine zipper is appended to proteins which originally form oligomers. Herein, we explored the control of the HRP-mediated protein heteroconjugation by introducing electric charges on the Y-tags. The electrostatic interaction between positive and negative charges clearly shows both attractive and repulsive forces between charges; therefore, the use of such interactions for conducting heteroconjugation is ideal. In addition, since the tyrosine-coupling reaction takes place through the formation of tyrosyl radicals, which have a very short lifetime, it can be expected that, if the attractive or repulsive interaction affects the accession of tyrosyl radicals, it will result in a significant difference in the cross-linking behavior. This was suggested from our previous study, where the negatively charged BAP possessing GY-tag was capable of co-cross-linking with the positively charged lysozyme, but it showed no reactivity against the negatively charged ovalbumin (OVA) or bovine serum albumin (BSA).22 Aspartic acid (Asp, D) and arginine (Arg, R) were used to electrostatically charge the Y-tags. The Y-tag sequences of DDDDY, DDYDD, RRRRY, and RRYRR, which are abbreviated as DY-tag, DYD-tag, RY-tag, and RYR-tag, respectively, were appended to the C-terminus of BAP and SA. We anticipated that the positive and negative charges of these Y-tags would hamper the self-cross-linking reaction while facilitating the heterocross-linking reaction between the oppositely charged Y-tags. The BAP mutants and SA mutants tagged with electrostatically charged Y-tags were treated by HRP, and the possibility of controlling the tyrosyl radical coupling reaction by electrostatic interaction was investigated.

(pET22b+ with gene coding BAP-GY in between its BamHI and XhoI sites). The gene coding the GY-tag and thrombin cleavage sequence (LVPRGSGGGGY) in the plasmid were substituted with genes coding an additional linker sequence and the positively or negatively charged Y-tags by site-directed mutagenesis using a KOD-Plus-Mutagenesis Kit as follows: DYtag (LVPRGSGGGSDDDDY), DYD-tag (LVPRGSGGGSDDYDD), RY-tag (LVPRGSGGGSRRRRY), and RYR-tag (LVPRGSGGGSRRYRR). The BAPs possessing these Y-tags are abbreviated as BAP-DY, BAP-DYD, BAP-RY, and BAPRYR, respectively. The additional linker sequence was added to ensure the thrombin recognition of the Y-tags. The constructed expression plasmids for BAP-DY, BAPDYD, BAP-RY, and BAP-RYR were transformed into E. coli BL21 (DE3), and the general expression and purification process for these BAP mutants were carried out as in our previous report,22 except in the purification step for BAP-RY and BAP-RYR using the anion exchange column, the pH value of the binding and the washing buffer was changed to 9.0. The BAP mutants were then purified using a SEC column equilibrated with the SEC mobile phase (10 mM Tris-HCl, pH 8.0, 150 mM NaCl), and finally desalted into 10 mM TrisHCl (pH 8.0) on a PD-10 column. The expression genes for SA mutants were prepared from the expression plasmid for SA-GY (pET22b+ with gene coding SAGY in between its NdeI and HindIII sites). The gene coding the GY-tag (GGGGY) in the plasmid was substituted to genes coding the DY-tag (DDDDY), DYD-tag (DDYDD), RY-tag (RRRRY), or RYR-tag (RRYRR) by site-directed mutagenesis. The SAs possessing these Y-tags are abbreviated as SA-DY, SADYD, SA-RY, and SA-RYR, respectively. The expression of these SA mutants was conducted using E. coli BL21 (DE3). The general expression and purification process of SA mutants was carried out as our previous report4 except the purification step was carried using an ion exchange column. For the purification of SA-RY and SA-RYR, a minor change in the procedure was made in the purification step using a cation exchange column due to the additional positive charges. The SA-RY and the SARYR were eluted from the column using a salt gradient of up to 75% of elution buffer (100 mM succinic acid, 10 mM EDTA, 1 M NaCl, pH 4.4). For SA-DY and SA-DYD, instead of using a cation exchange column, the SAs were purified using an anion exchange column and the purification condition for these SAs with the negatively charged Y-tags was the same as for the BAPs. The concentrations of BAPs and SAs were determined by a BCA assay with BSA as the standard. The purities of the prepared proteins were evaluated by SDS-PAGE analysis. Evaluation of the Effect of Negative and Positive Charges on Y-tags. The reactivity of newly prepared DY-tag, DYD-tag, RY-tag, and RYR-tag against HRP treatment was tested by treating BAP and SA mutants solely by HRP. BAP or SA and HRP were dissolved into 10 mM Tris-HCl (pH8.0) at a final concentration of 0.2 mg/mL and 0.1 mg/mL, respectively and H2O2 was then added to the solution 5 times every 10 min at a final concentration of 50 μM at 37 °C. The sample solution was incubated overnight at 37 °C and analyzed by SDS-PAGE analysis. To analyze whether the negative and positive charges on the Y-tags work to control the protein heteroconjugation, the SA mutants and various negatively charged proteins were treated together by HRP. The model conjugation partners for SA were EGFP(Y237F), OVA, and BSA. The EGFP(Y237F) was used because in our previous report, it was shown that the wild-type

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EXPERIMENTAL PROCEDURES Materials and Instruments. The wild-type BAP (WTBAP) and SA (WT-SA) and GY-tagged BAP (BAP-GY) and SA (SA-GY) were prepared according to our previous reports.4,22 HRP-type VI (250−330 units/mg solid using pyrogallol), albumin from chicken egg white grade V (OVA), and ribonuclease A from bovine pancreas (RNase A) were purchased from Sigma Aldrich Co. Hydrogen peroxide solution (30 wt %), lysozyme from chicken egg white, and albumin from bovine serum, cohn fraction V, pH 7.0 (BSA), were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). EGFP(Y237F) was prepared from the gene coding wild-type EGFP with an N-terminal His6-tag. The mutation was introduced by site-directed mutagenesis using a KOD-PlusMutagenesis Kit (Toyobo Co., Ltd. Life Science Department Osaka, Japan), and the expression and the purification of EGFP(Y237F) were done as previously reported.23 All other reagents were purchased and used as received. Prepacked columns including a Ni-NTA column (His-Trap HP column 5 mL), an anion-exchange column (HiTrap DEAE FF 5 mL), a cation exchange column (HiTrap SP HP 1 mL), and a desalting column (PD-10) were purchased from GE Healthcare BioSciences. A size-exclusion chromatography column was constructed by packing the XK16 column with Superdex 200 prep grade that were both purchased from GE Healthcare BioSciences. All of the chromatography experiments for the purification of BAPs and SAs were conducted on a BioLogic DuoFlow Chromatography System (Bio-Rad Laboratories, Inc.). Preparation of BAPs and SAs Possessing Negatively or Positively Charged Y-tags. The expression genes for BAP mutants were prepared from the expression plasmid of BAP-GY 1601

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mg/mL, respectively, in a total volume of 400 μL. The solution was placed into a quartz cell for fluorescence measurement (cell volume of 400 μL), and a portion of a 500 μM H2O2 solution was mixed into the solution to a final concentration of 50 μM to conduct the HRP treatment on the proteins. As soon as the reaction was started, the time-driven fluorescence measurement at 420 nm was initiated and the intensity was monitored for 3 min at 25 °C (excitation light wavelength of 315 nm, a sharpcut filter at 350 nm, both excitation and fluorescence slits of 10 nm). Investigation of the Function of the BAP-SA Conjugates on a Biotin-Coated Plate. BAP (0.2 mg/mL) and SA (0.2 mg/mL) were co-cross-linked by HRP treatment (0.1 mg/mL of HRP and 50 μM H2O2). After HRP treatment, the sample solutions were diluted using 10 mM Tris-HCl (pH 8.0) to a concentration of 0.5 unit/mL and 125 mM of NaCl was added. One hundred microliters of each BAP-SA conjugate solution was added to a well of a biotin-coated plate (Pierce Biotin Coated Clear 8-Well Strip Plates) preliminarily washed with TBS three times and then incubated for 30 min at 25 °C for immobilization. The wells were washed with TBS three times to remove unbound proteins. BAP enzymatic activity of the immobilized BAP-SA conjugates was measured by adding 200 μL of 1 mM p-NPP/1 M Tris-HCl (pH 8.0) to each well and the increase in the absorbance at 410 nm was monitored for 10 min at 37 °C. Two-Step Conjugation Reaction of BAP and SA. The BAP-SA conjugates were prepared by a two-step conjugation reaction. In the first step, H2O2 was added to a solution containing BAP and HRP twice at 37 °C to conduct the homoconjugation reaction of BAP. In the second step, additional HRP and SA were added to the solution and H 2 O 2 was added two more times to carry out the heteroconjugation reaction. The final concentration of BAP, SA, and HRP was 0.2 mg/mL and that of H2O2 was 50 μM. HRP was added twice because HRP may have been inactivated by H2O2 in the first step. The function of the BAP-SA conjugates prepared by the two-step conjugation reaction was evaluated by immobilizing the conjugates on the biotin-coated plate as described earlier. Investigation of Effect of Conjugation pH on the Function of BAP-SA Conjugates. The BAP-SA conjugates were prepared at various pH conditions from pH 5.0 to 9.0. Acetate buffer was used for the condition of pH 5.0. For pH 6.0 and 7.0, phosphatase buffer was used and Tris-HCl buffer was used to prepare the conditions of pH 8.0 and 9.0. All of the buffer concentrations were 50 mM. The preparation of BAP-SA conjugate was conducted the same as the one-step process described earlier. After conjugation reaction, all of the sample solutions were dialyzed against 10 mM Tris-HCl (pH 8.0) for 24 h at 4 °C using Slide-A-Lyzer Mini Dialysis Unit (7000 MWCO). The function of the BAP-SA conjugates prepared at different pH conditions was evaluated by immobilizing the conjugates on the biotin-coated plate as described earlier.

EGFP cross-linked by itself upon HRP treatment probably due to the tyrosine residue at its penultimate C-terminal position.22 SA mutants, model proteins, and HRP were mixed together in 10 mM Tris-HCl (pH 8.0) at final concentrations of 0.2 mg/ mL, 0.2 mg/mL, and 0.1 mg/mL, respectively. The heteroconjugation reaction was conducted by adding H2O2 in the same manner as described above, and the sample solutions were analyzed by SDS-PAGE. In addition, the BAP mutants were treated by HRP with the positively charged proteins, lysozyme and RNase A. The heteroconjugation reaction condition for BAP mutants and the model proteins was the same as for the heteroconjugation reaction between the SA mutants and various proteins described above. The samples were analyzed by SDS-PAGE analysis. SA mutants were not employed in this experiment, because SAs and the positively charged proteins have similar molecular weights, thus making it difficult to follow the cross-linking behavior by SDS-PAGE analysis. Heteroconjugation Reaction of BAP Mutants and SA Mutants. BAP mutants and SA mutants were treated together by HRP to examine the possibility of controlling the crosslinking between BAP and SA by the tethered positive or negative charges on the Y-tags. BAP and SA were dissolved into 10 mM Tris-HCl (pH 8.0) at a final concentration of ∼7 μM each, which is equivalent to 0.35 mg/mL and 0.1 mg/mL, respectively. The heteroconjugation reaction was initiated by adding H2O2 5 times every 10 min at a final concentration of 50 μM. The sample solutions were analyzed by SDS-PAGE and native-PAGE analyses. Prior to native-PAGE analyses, biotin-4fluorescein was added to the sample solutions at a final concentration of 50 μM and incubated for 1 h at 4 °C. Measurement of the Enzymatic Activities of BAP-SA Conjugates. After the heteroconjugation reaction described above, the sample solutions were diluted 10-fold by 10 mM Tris-HCl (pH 8.0), and 10 μL aliquots of the diluted solutions were dispensed into the wells of a 96-well microplate. To measure the BAP enzymatic activities of the BAP-SA conjugates, 190 μL of 1 mM p-nitrophenylphosphate (pNPP)/1 M Tris-HCl (pH 8.0) solution was added to each well, and the change in absorbance at 410 nm, derived from the formation of p-nitrophenol (p-NP) from p-NPP by BAP enzymatic activity, was monitored for 10 min at 37 °C. The enzymatic activities of untreated BAPs were also measured in the same manner and the obtained values were used to calculate the relative activity of each BAP-SA conjugate. To convert the enzymatic activity values measured on the microplate into unit/mg, the specific activities of untreated BAPs were measured using a UV−vis spectrometer in the following manner. Ten microliters of 0.1 mg/mL BAP solution was mixed with 990 μL of 1 mM p-NPP/1 M Tris-HCl (pH 8.0) in a UV quartz cell. The change in absorbance at 410 nm was monitored for 3 min at 25 °C. One unit of BAP enzymatic activity was defined as the activity that catalyzes the dephosphorization reaction of 1 μmol of p-NPP in 1 min where the p-NP absorbance coefficient of 16 900 M−1 cm−1 was employed to calculate the unit value. Fluorescence Measurement of Dityrosine. The reactivities of the RY-tag and RYR-tag against HRP treatment was evaluated by measuring the fluorescence intensity increase at 420 nm derived from dityrosine formation. The fluorescence measurement was carried out on a Perkin-Elmer LS55. Either SA-RY or SA-RYR and HRP were dissolved into 10 mM TrisHCl (pH 8.0) at a final concentration of 0.2 mg/mL and 0.1

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RESULTS AND DISCUSSION Reactivity of the Negatively or Positively Charged Ytags against HRP Treatment. It has been demonstrated by our previous work that the GY-tag, a very flexible peptide sequence consisting of GGGGY, is efficiently recognized by HRP resulting in the conjugation of the GY-tagged proteins to each other. In contrast, DY-tag, DYD-tag, RY-tag, and RYR-tag are much more sterically hindered at the tyrosine residue 1602

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reaction site and also have strong negative or positive charges; therefore, it seemed that the tyrosine residues in these tags were much less likely to be recognized by HRP. Figure 1 shows the result of the SDS-PAGE analysis of BAP mutants and SA mutants after HRP treatment.

As shown in lane 2 for each protein, RY- and RYR-tagged BAP and SA were cross-linked upon HRP treatment resulting in the disappearance of bands for mBAP and mSA. Thus, despite the surrounding positive charges and steric hindrance caused by arginine, the tyrosine residue in either RY-tag or RYR-tag can be recognized by HRP. The homoconjugation reaction of BAP-GY primarily produced a mBAP dimer as visualized by SDS-PAGE analysis (lane 2 for BAP-GY). Interestingly, from the results shown in lanes 2 for the BAPs with the positively charged Y-tags (BAP-RY and BAP-RYR), almost no mBAP dimer was observed after HRP treatment and the band intensity for the mBAP polymers significantly increased compared with BAP-GY. The difference of crosslinking behavior between BAP-GY and BAP-RY or BAP-RYR suggests that BAP-RY and BAP-RYR cross-linked via some of their intrinsic tyrosine residues. BAP itself has a relatively low pI value, ∼4.5,24 so the BAP surface charges are strongly negative under the reaction conditions at pH 8.0. Meanwhile, RY- and RYR-tags have positive charges within a small region. Therefore, RY- and RYR-tags electrostatically interacted with BAP itself and cross-linked with the intrinsic tyrosine residues on BAP, resulting in complex conjugates which appeared as polymers on SDS-PAGE. In contrast, in the case of SA, we could not see a clear difference in reactivity between the positively charged Y-tags and GY-tag, all resulting in the formation of broad, blurred bands of mSA polymer. This is probably because SA has several intrinsic tyrosine residues that are largely exposed on its surface which can react with GY-tag.4 DY- and DYD-tagged BAP and SA showed much lower reactivity compared with other mutants against HRP treatment

Figure 1. SDS-PAGE analyses of the homoconjugation reaction of BAP mutants (A) and SA mutants (B). Lane 1: SA or BAP; Lane 2: SA or BAP with the addition of HRP and H2O2.

Figure 2. SDS-PAGE analyses of the heteroconjugation reaction of negatively charged proteins with SA mutants (A) and positively charged proteins with BAP mutants (B). All lanes: HRP and H2O2 were added. Lane 1: each protein; Lane 2: each protein with the addition of GY-tagged SA or BAP; Lane 3: each protein with the addition of RY-tagged SA or BAP; Lane 4: each protein with the addition of RYR-tagged SA or BAP; Lane 5: each protein with the addition of BAP-DY; Lane 6: each protein with the addition of BAP-DYD. The red arrows indicate bands corresponding to heteroconjugates of each protein with mSA or mBAP. 1603

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Table 1. Properties of Proteins Used in this Studya proteins

M.W.

pI

Tyr

self-cross-linking upon HRP treatment

WT-BAP BAP-GY BAP-DY or -DYD BAP-RY or -RYR WT-SA SA-GY SA-DY or -DYD SA-RY or -RYR EGFP(Y237F) BSA OVA Lysozyme RNase A HRP

48 809.48b 49 810.59b 50 300.97b 50 465.37b 13 402.62b 13 794.00b 14 026.15b 14 190.54b 27 879.52b 67 kDa 45 kDa 14 kDa 14 kDa 44 kDa

4.524 5.73b 5.44b 6.06b 5−632 6.04b 4.74b 9.34b 4.6−5.425 4.626 4.627 9.328 9.629 9.433

11 12 12 12 6 7 7 7 10 20 10 3 6 5c

ND High (cross-linking is selective at GY-tag) Low (inhibited by electrostatic repulsion) High (intrinsic Tyr residues are involved) Low (Y72 is recognized by HRP4) High (intrinsic Tyr residues are involved) Low (inhibited by electrostatic repulsion) High (intrinsic Tyr residues are involved) ND ND ND ND ND ND

a M.W.: molecular weight of monomer; ND: not detected; Tyr: number of tyrosine residues in a single polypeptide chain. bCalculated values using a web tool, Compute pI/Mw, on ExPASy server at http://web.expasy.org/compute_pi/. cBased on the sequence of HRP C1A (PDB:1H58).

corresponding bands for the heteroconjugates were not clearly observed in lanes 3 and 4 for lysozyme where BAP-RY and BAP-RYR were co-cross-linked, respectively. These results indicate that the positive charges on the RY- and RYR-tags inhibited the approach of the Y-tag to the positively charged lysozyme. RNase A co-cross-linked with all of the BAP mutants, resulting in a band for the heteroconjugate at around 60 kDa (lanes 2, 3, and 4 for RNase A in Figure 2B). Yet, the intensity of the bands decreased in BAP-RY and BAP-RYR compared with BAP-GY, suggesting the inhibitive effect of cross-linking with RNase A. The positive charge of the RNase A is concentrated mainly on the surface of its RNA binding site while the remainder is not so highly positively charged (PDB ID: 2AAS). In contrast, the positive charges are widely distributed on the whole surface of lysozyme (PDB ID: 1LYS). We assumed that the differences in the charge distribution on the protein surface between RNase A and lysozyme caused the differences in the cross-linking behaviors. Intriguingly, as shown from the results of lanes 5 and 6 for both lysozyme and RNase A, BAP-DY and BAP-DYD were cocross-linked with these positively charged proteins with considerably high efficiency compared with BAP-GY (lane 2). When the DY- and DYD-tagged BAP and SA were treated by HRP alone, almost no self-cross-linked products were observed. Thus, it can be concluded that, in the homoconjugation reaction of DY- and DYD-tagged BAPs and SAs, the DY- and DYD-tags were indeed recognized by HRP in the solution, but they repelled each other, and the negatively charged BAP and SA resulting in suppression of the self-cross-linking. It was also interesting to understand how the activated DY- and DYD-tags act under conditions where there is no cross-linking partner. It is possible that the activated negatively charged Y-tags are repaired by the reaction with superoxide, 30 which can be generated by the alternative catalytic cycle of HRP.31 They may also react with proteinaceous impurities in the reaction mixture, which is suggested by the formation of very weak broad bands in the high-molecular-weight region in lanes 2 for BAP-DY and BAP-DYD in Figure 1; however, further study is needed to clarify this point. The properties of all of the proteins used in this study are summarized in Table 1. From the results obtained, it was clearly shown that the negative and positive charges on the Y-tags definitely alter the cross-linking behavior of Y-tagged proteins,

as observed by the intact bands for mBAP and mSA after HRP treatment. Since the number of carbons in the side chain of aspartic acid is smaller than that of arginine, the effect of the steric hindrance on HRP recognition of the tyrosine residue can be excluded as a possible reason for the low reactivity. Alternatively, it is possible that the negative charges hampered the HRP recognition. The reactivity of DY-tag and DYD-tag will be discussed further in subsequent sections. Heteroconjugation Reaction of BAP and SA Mutants with Various Proteins. Next, to further investigate the crosslinking behavior of reactive RY- and RYR-tags, heteroconjugation reactions of SA-RY and SA-RYR with negatively charged proteins, EGFP(Y237F) (pI: 4.6−5.4),25 BSA (pI: 4.6),26 and OVA (pI: 4.6),27 were conducted, and the results of SDS-PAGE analyses are shown in Figure 2. In lanes 3 and 4 for each protein in Figure 2A, bands corresponding to the heteroconjugates of mSA with each protein emerged after HRP treatment. The corresponding bands were not observed in lanes 2 for BSA and OVA, where the band for co-cross-linked SA-GY was identified. EGFP(Y237F) has several tyrosine residues that are largely exposed on its surface; therefore, it formed heteroconjugates with SA-GY. However, the band intensity of the heteroconjugate was weaker than that observed in lanes 3 and 4. Taken together with the results of the homoconjugation reaction of BAP-RY and BAP-RYR (Figure 1A), it can be concluded that the protein heteroconjugation reaction was directed to the negatively charged proteins by the positively charged RY-tag and RYR-tag. Furthermore, RY- and RYR-tagged BAP mutants were treated by HRP in the presence of the positively charged proteins lysozyme (pI: 9.3)28 and RNase A (pI: 9.6), 29 to determine the reactivity of RY- and RYR-tags against positively charged proteins. Also, BAP-DY and BAP-DYD were employed in this experiment to identify whether the low reactivity of the negatively charged Y-tags against HRP treatment was because the negative charges inhibit the recognition of HRP or the negative charges repel each other, hence hampering the crosslinking of tyrosyl radicals. As shown in lane 2, the BAP-GY cross-linked with the lysozyme after HRP treatment because of some exposed tyrosine residues on lysozyme and the electrostatic interaction between positively charged lysozyme and negatively charged BAP itself, resulting in a band for the mBAP-lysozyme conjugate at around 50 kDa. In contrast, 1604

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and it was largely affected by the charge on the protein surfaces. Since the model proteins studied here, BAP and SA, are both negatively charged under the reaction conditions of pH 8.0 (pI values for BAP and SA are 4.5 and 5−6,32 respectively), the RYand RYR-tags resulted in random self-cross-linking of BAP or SA upon HRP treatment, but they inhibited the co-crosslinking of BAP against positively charged proteins. The negative charges on the DY- and DYD-tags almost completely suppressed the self-cross-linking of BAP and SA, but the BAP with the negatively charged Y-tags showed co-cross-linking ability against positively charged proteins. From these results, two conclusions were drawn. One is that the RY- and RYR-tags were mainly cross-linked to the intrinsic tyrosine residues of BAP and SA, not to the tyrosine residue in the RY- and RYRtags. Second is that, if the RY- and RYR-tags were tethered to positively charged proteins, they might show reactivity as low as that of the DY- and DYD-tags obtained in this study. One interesting fact is that the electrostatic interaction between HRP and those positively or negatively charged Y-tags showed no apparent effect on the cross-linking of Y-tagged proteins. The exact pI value of HRP used in this study is not clear since HRP exists as a group of isozymes. However, the pI value for one of the major isozymes, HRP C1, was reported to be 9.4,33 and also, the HRP passes through an anion-exchange column equilibrated with a buffer of pH 8.0;22 it can be predicted that the HRP used in this study was positively charged in the reaction condition. Even though HRP is positively charged, the DY- and the DYD-tagged proteins showed no reactivity against HRP. The inertness of HRP against Y-tag mediated protein cross-linking is probably due to the glycosylation on HRP, by which sugar chains might act like shields against the radical species that HRP generates. Also, the positively charged Y-tags were efficiently recognized by positively charged HRP. This result indicates that the electrostatic repulsion may not dominate the substrate recognition of HRP. The results showed that the cross-linking of Y-tagged proteins was significantly affected by the electrostatic interaction between substrate proteins. This can be explained as follows. The HRP mediated protein cross-linking reaction using Y-tags is initiated by formation of tyrosyl radicals. As the tyrosyl radicals have a short lifetime, the subtle repulsive or attractive interaction between Y-tags and proteins showed major impact on the tyrosyl radical coupling reaction. That is to say, the cross-linking of Y-tagged proteins was controlled by the spatial-temporal manner in which tyrosyl radical accession is regulated by the electrostatic interaction. Heteroconjugation Reaction between BAP Mutants and SA Mutants. Next, BAP mutants and SA mutants were co-cross-linked with each other to investigate the cross-linking properties between electrostatically charged Y-tags. The representative results of the SDS-PAGE and native-PAGE analyses for the heteroconjugation reaction between WT-BAP, BAP-GY, BAP-DYD, or BAP-RYR and the SA mutants are shown in Figure 3 (see Figure S1 and Figure S2 in the Supporting Information for more results). From lanes 3 and 4 for BAP-GY in Figure 3A, where SA-RY and SA-RYR were co-cross-linked, respectively, the band intensity of the mBAP dimer at 100 kDa significantly decreased while the band intensity of the polymers increased, compared with lane 1 where SA-GY was co-cross-linked. The differences in the resulting bands were caused by the following reason (Figure 4). The BAP-GY cross-links by itself through its GY-tag

Figure 3. SDS-PAGE analyses (A) and native-PAGE analyses (B) of the heteroconjugation reaction of BAP-GY with SA mutants. All lanes: HRP and H2O2 were added; Lane 1: BAP with the addition of SA-GY; Lane 2: BAP with the addition of SA-DY; Lane 3: BAP with the addition of SA-RY; Lane 4: BAP with the addition of SA-RYR. In the native-PAGE analysis, SA was visualized by biotin-4-fluorescein (right of each picture).

mostly in a linear form,22 which appears as mBAP dimers in SDS-PAGE analysis (lane 2 for BAP-GY in Figure 1A and Figure 4A). Meanwhile, the positively charged Y-tags are capable of cross-linking with the intrinsic tyrosine residues of BAP, resulting in mBAP polymers (lane 2 for BAP-RY and BAP-RYR in Figure 1A and as depicted in Figure 4B). Also, in lanes 3 and 4 in Figure 3B, where SA-RY and SA-RYR were cross-linked with BAP-GY, a portion of the resultant BAP-SA conjugates barely entered the stacking gel, while most of the (BAP-GY)-(SA-GY) heteroconjugate penetrated through the stacking gel. The results obtained here indicate that the SAs with positively charged Y-tags directed the cross-linking of SAs toward negatively charged BAP-GY and favorably cross-linked not only with the GY-tag, but also with the intrinsic tyrosine residues, yielding less mBAP dimer and more mBAP-mSA polymers (Figure 4C). BAP possesses three tyrosine residues that are located near the protein surface and are suspected to be the cross-linking site with the positively charged Y-tags (Figure S3 in the Supporting Information). Meanwhile, SA has several largely exposed tyrosine residues, which can be cross-linked even with the GY-tag. In the heteroconjugation reaction between BAP-GY and SA with positively charged Y-tags, the 1605

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Enzymatic Activities of BAP-SA Conjugates. To investigate the effect of the cross-linking using the SAs with positively charged Y-tags, the BAP enzymatic activities of the BAP-SA conjugate were measured after the heteroconjugation reaction. WT-BAP heteroconjugates co-cross-linked with SARY and SA-RYR formed precipitates after HRP treatment; therefore, measurement of the enzymatic activity of these heteroconjugates was not possible. The results for the (WTBAP)-(SA-GY) conjugates and (BAP-GY)-SA conjugates are shown in Figure 5. All of the BAP-SA conjugates retained more

Figure 4. Schematic illustration of the possible forms of cross-linked BAP and SA. (A) Self-cross-linking of BAP-GY. (B) Self-cross-linking of BAP-RY or BAP-RYR. (C) Co-cross-linking of BAP-GY with SA-RY or SA-RYR. Only representative forms of cross-linking are illustrated.

intrinsic tyrosine residues of BAP are involved in cross-linking with the positively charged Y-tags. In such conditions, both BAP and SA have multiple cross-linking sites in each polypeptide, leading to formation of highly complex heteroconjugates (Figure 4C). This can be supported by the results in lanes 3 and 4 for WT-BAP in Figure 3A, where SA-RY and SARYR were capable of cross-linking with WT-BAP, resulting in mBAP-mSA bands around 60 kDa. In comparison with the combination of BAP-GY and SA-GY, the overall BAP-GY crosslinking yield was increased when BAP-GY and SA-RYR was cocross-linked (Figure S4 in the Supporting Information). This result also supports the favorable cross-linking formation of RYR-tag with BAP. In addition, when WT-BAP was co-crosslinked with the SAs with positively charged Y-tags, precipitates were formed in the solution after HRP treatment. Precipitates were not observed for BAP-GY, suggesting that the GY-tag is actively involved in cross-linking with the RY- or RYR-tag to prevent the intrinsic tyrosine residues of BAP from crosslinking. In Figure 3A, lane 2, as shown for BAP-GY with the addition of SA-DY, in spite of the presence of reactive BAP-GY in the system, the mSA band remained and the profiles of the resulting bands of conjugates are almost same as that of WT-SA (Figure S1 and Figure S2 in the Supporting Information), suggesting that the negative charges on the DY-tag repelled the SAs from BAP. When SAs with negatively charged Y-tags were co-cross-linked with BAP-RY or BAP-RYR, the negative charges worked to direct the cross-linking of SAs toward BAP, resulting in the formation of bands for mBAP-mSA as observed in SDSPAGE analysis (lane 2 for BAP-RYR in Figure 3A). Similar results were obtained when the SAs with positive Y-tags and BAPs with negative Y-tags were co-cross-linked (lane 4 for BAP-DYD in Figure 3A). From these results, it was concluded that the charges on the Y-tags interact not only with the charges of the protein surfaces but also with the charges on the Y-tags.

Figure 5. Enzymatic activity of BAP-SA conjugates. The enzymatic activities of untreated WT-BAP and BAP-GY were defined as 100% of the relative activity of each BAP.

than 80% of their enzymatic activities. The negative effect of cocross-linking with positively charged Y-tags on the enzymatic activity was alleviated for the BAP-GY heteroconjugates. By comparison, the heteroconjugation reaction of RY- or RYRtagged BAPs with SA mutants significantly decreased their enzymatic activities by about 40%, probably because of intramolecular cross-linking of the RY- and RYR-tags onto BAP’s intrinsic tyrosine residues (Figure S6 in the Supporting Information). When the SA-DY and SA-DYDs were co-crosslinked, the reduction in the enzymatic activity of BAP-RY and BAP-RYR was relatively small, although the overall heteroconjugation efficiency was not high, probably due to intramolecular cross-linking of BAPs with positively charged Y-tags (Figure S2 in the Supporting Information). In Figure 3B, it is clearly seen that the BAP-GY was highly cross-linked to form extremely complex heteroconjugates with SAs with retaining the biotin binding ability. It is amazing that even in such a complex highly cross-linked form, the (BAP-GY)-SA conjugates substantially retained the BAP enzymatic activity, demonstrating the advantage of tethering the GY-tags to BAP. Kinetically Controlled Tyrosine Radical Coupling Reaction. The reactivities of RY- and RYR-tags were evaluated by measuring the dityrosine fluorescence change upon HRP treatment (Figure 6). The fluorescence intensity for SA-RY increased rapidly, implying that the formation of dityrosine was completed within a short time period. The fluorescence intensity of SA-RYR increased slowly within the time period of measurement. Since the tyrosine residue in the RYR-tag is encompassed by positively charged arginines, the steric hindrance and the effect of the positive charges on the tyrosine 1606

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Figure 6. Time-driven measurements of fluorescence intensity of dityrosine at 420 nm.

residue are maximized. Therefore, the recognition of the tyrosine residue by HRP is inhibited, resulting in slow dityrosine formation. The results suggested that heteroconjugation can be kinetically controlled. When focusing on the molar ratio of the protein components of BAP-SA conjugates, the heteroconjugates which contain a higher ratio of BAP to SA are likely to perform well for the purposes of detecting a biotin moiety immobilized on a solid surface. To control the conjugation ratio between (BAP)n and (SA)m as n > m, we attempted to kinetically control the BAPSA heteroconjugation using BAP-GY and either SA-GY or SAs with positively charged Y-tags. The characteristics of heteroconjugates obtained under different conditions were compared by BAP enzymatic activity on a biotin-coated plate (Figure 7). The BAP-SA conjugates prepared using SA-RY or SA-RYR showed higher enzymatic activity than the (BAP-GY)-(SA-GY) conjugate (Figure 7A). In particular, (BAP-GY)-(SA-RYR) performed the best in this experiment, increasing the BAP enzyme activity by 40% of the conjugate using SA-GY. The BAP enzymatic activities of all samples were adjusted to 0.5 U/ mL before immobilizing the BAP-SA conjugate on the biotincoated plate, and the BAP enzymatic activity of each (BAPGY)-SA conjugate measured in the solution state was essentially identical (Figure 5). Hence, the variations in the BAP enzymatic activities measured on the plate represent the differences in the BAP-SA conjugation ratio. The GY-tag gives BAP the ability to intermolecularly cross-link through the Y-tag while minimizing the unfavorable intramolecular cross-linking between the GY-tag and the intrinsic tyrosine residues. The RYR-tag shows slow reactivity upon HRP treatment providing more opportunities for BAP-GY to yield (BAP-GY) n homoconjugates,22 and it also shows favorable cross-linking formation toward negatively charged BAP-GY due to its positive charges. As a result, the combination of BAP-GY and SA-RYR worked well to obtain the (BAP-GY)n-(SA-RYR)m conjugate of which n > m. The results described herein indicated that, by introducing the GY-tag to BAP and the RYRtags to SA, the heteroconjugation reaction between BAP and SA was kinetically controlled. To test this idea further, we performed a two-step conjugation reaction where BAP-GY was first pretreated by HRP to form (BAP-GY)n homoconjugates, then SAs were cocross-linked toward (BAP-GY)n. Figure 7B clearly illustrates the advantage of SA-RY and SA-RYR over SA-GY, as the conjugation partner for BAP-GY. The BAP-SA conjugate prepared by using SA-RY and SA-RYR showed about double and triple activity of the conjugate of SA-GY, respectively, and notably, the (BAP-GY)-(SA-RYR) conjugate showed quite

Figure 7. Enzymatic activity of the BAP-SA conjugates on a biotincoated plate. (A) BAP-SA conjugates prepared by one-step conjugation reaction; (B) BAP-SA conjugates prepared by two-step conjugation reaction.

similar enzymatic activity to that prepared by the single step. In the reaction system, many types of cross-linking are taking place such as intramolecular cross-linking of Y-tags or Y-tags with intrinsic tyrosine residues and intermolecular cross-linking between (BAP-GY)n-(BAP-GY)n, (BAP-GY)n-SA, and SA-SA. It can be considered that the intermolecular cross-linking between (BAP-GY)n and SA competes directly with the crosslinking between SA-SA, since the cross-linking between (BAPGY)n-(BAP-GY)n seems negligible due to the low concentration of (BAP-GY)n. In Figure 7B, the SAs with positively charged Y-tags had better enzymatic activity than the SA-GY in the two-step conjugation, indicating that the RY- and RYR-tags have a higher tendency to cross-link with (BAP-GY)n than the GY-tag. The SA-RYR showed higher performance than the SARY because the self-cross-linking rate of the RYR-tag is lower than that of RY-tag (Figure 6), indicating less SA-SA crosslinking. Last, the effect of the conjugation pH on the activity of BAPSA conjugates was evaluated. The BAP-SA conjugates were prepared with different pairs of BAP and SA mutants at different pH, and the enzymatic activity was measured after immobilization on the biotin-coated plate (Figure 8). The BAP1607

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In conclusion, by controlling the intramolecular and intermolecular cross-linking rates by means of tethering appropriate electrostatic interactions onto the Y-tags and also optimizing the Y-tag sequence, it is possible to kinetically control the heteroconjugation reactions of distinct proteins via a tyrosine radical-coupling reaction to maximize a specific functionality of the protein heteroconjugates.

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CONCLUSION The cross-linking of electrostatically charged Y-tags was strongly dependent on the charges of the protein surfaces. The DY- and DYD-tags showed a considerably high crosslinking efficiency against positively charged protein, while the DY- and DYD-tagged BAP and SA were almost inactive in terms of self-cross-linking. In contrast, the RY- and RYR-tagged BAP and SA were efficiently self-cross-linked and also co-crosslinked with negatively charged proteins. In the heteroconjugation reaction of BAP and SA, SAs possessing positively charged Y-tags favorably co-cross-linked with negatively charged BAP with GY-tag. The GY-tag on BAP had high self-cross-linking ability while retaining BAP’s enzymatic activity. When comparing the reactivity of RY- and RYR-tags, the RYR-tag formed dityrosine pairs slower than the RY-tag. Taking all the results into consideration, it was concluded that the BAP-SA conjugation pair which had the best performance on the biotincoated plate was BAP-GY and SA-RYR. The (BAP-GY)-(SARYR) conjugate showed higher enzymatic activity on a biotincoated plate compared with its counterpart, the (BAP-GY)(SA-GY) conjugate. From our results, it can be concluded that by introducing very simple electrostatic interactions between protein and Y-tags and by optimizing the HRP recognition reactivity of Y-tag it was possible to kinetically control the heteroconjugation of proteins via a tyrosyl-radical coupling reaction.

Figure 8. Effect of the conjugation pH on the function of BAP-SA conjugates on a biotin-coated plate. The measured activity of (BAPGY)-(SA-GY) conjugate prepared at pH 8.0 was defined as 100%. pH 5.0: acetate buffer; pH 6.0 and pH 7.0: phosphate buffer; pH 8.0 and pH 9.0: Tris-HCl buffer.

SA conjugates prepared at pH 6.0 and 7.0 showed high enzymatic activity for all combinations of BAP and SA. Since the pI value of SA is close to neutral, the repulsive interaction between BAP-GY and SA-GY is minimized at pH 6.0 and 7.0, resulting a higher cross-linking efficiency compared with higher pH conditions where SA-GY is negatively charged. The advantages of positive charges on RY-tag and RYR-tag over GY-tag still can be observed at these pH conditions, probably because the positive charges on Y-tag act to hamper the selfcross-linking of SA mutants while facilitating the cross-linking toward negatively charged BAP-GY. In addition, HRP has its optimal condition at pH 6.0−6.5; therefore, the Y-tags were efficiently activated by HRP at this pH range. At pH 5.0, the activity of all of BAP-SA conjugates was decreased, probably due to lower HRP activity. Intriguingly, the (BAP-GY)-(SARYR) conjugates prepared at the conjugation pH values of 8.0 and 9.0 showed high activity, as well as at pH 6.0 and 7.0. The other BAP-SA conjugates decreased the activity on the plate at pH 8.0 and 9.0. The conjugation pH independency of (BAPGY)-(SA-RYR) conjugate can be explained by the reduced tyrosine radical formation rate of RYR-tag. From the results obtained here, it can be concluded that, in the tyrosyl radical mediated protein cross-linking reaction, both the rate of tyrosyl radical formation and the coupling reaction rate are important to the heteroconjugation. The position of tyrosine residue in the Y-tag affects the tyrosyl radical formation, i.e., HRP recognition. The positive and negative charges on Y-tags affect mainly the tyrosyl radical coupling reaction, i.e., the reaction takes place after HRP recognition. For comparison, in the enzyme-mediated protein heteroconjugation catalyzed by MTG or SrtA,13−15 The cross-linking process is strictly regulated by substrates recognition by these enzymes, and the cross-linking efficiency is mostly dependent on whether the peptide sequence is suitable or not. Therefore, it can be said that controlling the protein coupling reaction by using such electrostatic interaction is unique to the tyrosyl radical-mediated protein cross-linking. The insights obtained in this study will be helpful for further application of the protein cross-linking method using Y-tag and HRP.

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ASSOCIATED CONTENT

S Supporting Information *

Heteroconjugation reaction between BAP mutants and SA mutants. The intrinsic tyrosine residues of BAP and SA, which are suspected to be involved in cross-linking. Study of crosslinking efficiency of BAP in the heteroconjugation reaction between BAP and SA. Thrombin treatment reaction on BAPSA conjugates. The enzymatic activity of BAP-RY and BAPRYR after heteroconjugation reaction with SA mutants. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +81-92-802-2806. FAX: +81-92-802-2810. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by a Grant-in-Aid for the Global COE program, “Science for Future Molecular Systems” from the Ministry of Education, Culture, Sports, Science and Technology of Japan. K.M. was supported by Research Fellowships of the Japan Society for the Promotion of Science (JSPS) for Young Scientists. 1608

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ABBREVIATIONS BAP, bacterial alkaline phosphatase; BSA, bovine serum albumin; EDTA, ethylenediaminetetraacetic acid; EGFP, enhanced green fluorescent protein; SA, streptavidin; NPP, nitrophenylphosphate; HRP, horseradish peroxidase; m, monomeric; NP, nitrophenol; OVA, ovalbumin; RNase, ribonuclease; SEC, size-exclusion chromatography; SDSPAGE, sodium dodecyl sulfate poly acrylamide gel electrophoresis; Tris, tris(hydroxymethyl)aminomethane

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