Supporting Information Site-specific protein cross-linking by peroxidase-catalyzed activation of a tyrosine-containing peptide tag Kosuke Minamihata 1, Masahiro Goto 1,2, Noriho Kamiya 1,2* 1 Department of Applied Chemistry, Graduate School of Engineering, Kyushu University 2 Center for Future Chemistry, Kyushu University * Corresponding author: Dr. Noriho Kamiya Phone: +81-92-82-286 FAX: +81-92-82-281 E-mail: nori_kamiya@mail.cstm.kyushu-u.ac.jp CONTENTS 1. EXPERIMENTAL PROCEDURES 2. SUPPORTING FIGURES AND TABLES 3. REFERENCE 1
1. EXPERIMENTAL PROCEDURES Materials. Molecular weight markers for SEC analysis (Gel Filtration Calibration Kit HMW) were purchased from GE Healthcare Bio-Sciences. Albumin bovine Cohn Fraction V, ph 7. (BSA), albumin from chicken egg (OVA), and avidin and lysozyme from egg white were purchased from Wako Pure Chemicals Industries. An Enhanced green fluorescent protein (EGFP) bearing an N-terminal hexahistidine tag was prepared according to a previous report (S1). Effect of reaction conditions on the cross-linking of Y-tagged BAPs. Cross-linking reactions of Y-tagged BAPs were conducted by changing HRP and H 2 O 2 concentrations. The BAP concentration was fixed at.2 mg/ml in 1 mm Tris-HCl (ph 8.) and H 2 O 2 was added 1 times at 1 min intervals. After the reactions were completed the samples were analyzed by SDS-PAGE. The effects of HRP and H 2 O 2 concentrations on the cross-linking of Y-tagged BAPs are shown in Figures S1 and S2, respectively. In addition, the effect of divided injections of H 2 O 2 on Y-tag BAP cross-linking was investigated. BAP, HRP and H 2 O 2 concentrations were fixed at.2 mg/ml,.1 mg/ml and 5 µm, respectively, and the cross-linking reaction of Y-tagged BAPs was conducted by varying the number of H 2 O 2 injections. The results of SDS-PAGE analysis of the sample solutions are presented in Figure S3. Estimation of the molecular weight of the cross-linked BAPs. Molecular weight markers for SEC analyses were prepared by dissolving ovalbumin at 4. mg/ml, conalbumin at 3. mg/ml, aldolase at 4. mg/ml and ferritin at.3 mg/ml in 5 mm Tris-HCl (ph7.2), 15 mm NaCl. Blue dextran was separately dissolved in the same buffer at a concentration of 1. mg/ml. The molecular weight markers and blue dextran solutions were applied to the SEC column (Superdex 2 1/3 GL) with an injection volume of 1 µl. The flow rate of the mobile phase (5 mm Tris-HCl, 15 mm NaCl, ph 7.5) was set at.2 ml/min and the absorbance of the eluate was monitored at 28 nm. Using the resultant chromatograms, the elution volumes (V E ) of each protein and blue dextran were estimated and K av values were calculated, where K av = (V E V O )/(V C V O ), V E : elution volume; V O : void volume (V E for blue dextran); V C :column volume (24 ml for the SEC column used). The V E and K av values of the molecular weight marker proteins and blue dextran are shown in Table S1. The obtained chromatogram of the molecular weight markers and blue dextran and the standard curve for the molecular weight are presented in Figure S4. V E and K av values of the cross-linked BAPs numbered in Figure 3 in the R&D section are listed in Table S2 and the deduced molecular weight of each peak is shown in Figure S5. 2
Co-cross-linking reaction of various proteins and Y-tagged BAPs. BSA, OVA, avidin, lysozyme and EGFP were co-cross-linked with the Y-tagged BAP by HRP treatment. The concentrations of each protein and the Y-tagged BAPs were set at.2 mg/ml. The HRP and H 2 O 2 concentrations were.1 mg/ml and 5 µm, respectively, and H 2 O 2 was added 1 times as divided injections. The sample solutions were analyzed by SDS-PAGE following the completion of the cross-linking reaction. The results are shown in Figure S6. BSA and OVA have pi values similar to BAP, therefore BAP and these proteins are both negatively charged in the reaction condition used (ph 8.). Although the crystalline structure of BSA is not available, matured BSA bears 2 tyrosine residues and some of them may be exposed on the surface. Additionally, OVA has 1 tyrosine residues and some tyrosine residues are exposed on the surface (PDB ID: 1OVA). However, BSA and OVA did not react with Y-tagged BAPs, indicating that an electrostatic repulsion between Y-tagged BAPs and these two proteins inhibited the access of the tyrosyl radicals of the Y-tag to the intrinsic tyrosine residues in BSA and OVA. The use of lysozyme resulted in co-cross-linking with the Y-tagged BAPs, with clearly apparent bands in the SDS-PAGE at ~76 kda. Lysozyme has a pi value of ~11 and hence in the reaction conditions, BAP and lysozyme have opposite charges, negative and positive, respectively. Lysozyme itself was not affected by HRP treatment (see lane 2 in the gel run for the lysozyme reaction); although it has exposed tyrosine residues (PDB ID: 1LYS). However, since there is no electric repulsion force between the Y-tagged BAP and lysozyme, the generated tyrosyl radical in the Y-tag can interact with the intrinsic tyrosine residues on the surface of lysozyme to exchange the radical and further react to give the lysozyme-bap conjugate. Like lysozyme, avidin possesses a high pi value (1.5), however avidin did not react with the Y-tagged BAP. Avidin has only one tyrosine residue in one unit and the tyrosine residue is oriented away from the surface of the protein (PDB ID: 1AVD). Therefore, the tyrosyl radical in the Y-tag could not access the intrinsic tyrosine residue in avidin. Consequently, no co-cross-linking product was observed. Lastly, EGFP, whose pi value is 5.7, cross-linked by itself upon HRP treatment and also co-cross-linked with the Y-tagged BAP. EGFP (PDB ID: 1GFL) has a flexible C-terminal chain and a tyrosine residue is located at penultimate C-terminal position. As such, EGFP already possesses a Y-tag and this C-terminal tyrosine residue is probably responsible for the self-cross-linking and co-cross-linking with the Y-tagged BAP upon HRP treatment. In summary, protein cross-linking reactions via the oxidative tyrosine coupling reaction is strongly dependent on the surface charge of the proteins, the orientation of tyrosine residues and 3
in particular the presence of flexible tyrosine residues, i.e., Y-tag. By controlling the reaction conditions such as the ph and salt concentration, it might be possible to conduct hetero-conjugation of proteins using oxidative tyrosine residues. 4
2. SUPPORTING FIGURES AND TABLES Figure S1. The effect of HRP concentrations on the cross-linking of Y-tagged BAPs studied by SDS-PAGE. Lanes 1 5: HRP concentrations of.1,.25,.5,.1 and.2 mg/ml. The concentrations of BAP and H 2 O 2 were.2 mg/ml and 5 µm, respectively. 5
Figure S2. The effect of H 2 O 2 concentrations on the cross-linking of Y-tagged BAPs studied by SDS-PAGE. Lanes 1 6: H 2 O 2 concentrations of 2.5, 5, 1, 25, 5 and 1 µm. The concentrations of BAP and HRP were.2 mg/ml and.1 mg/ml, respectively. 6
Figure S3. The effect of the number of divided H 2 O 2 injections on the cross-linking of Y-tagged BAPs studied by SDS-PAGE. Lanes 1 6: H 2 O 2 was injected once, twice, four, six, eight and ten times. The concentrations of BAP, HRP and H 2 O 2 were.2 mg/ml,.1 mg/ml and 5 µm, respectively. 7
Table S1. Results of the SEC analysis of the molecular weight markers and blue dextran Contents MW [Da] V E [ml] K av 1 Ferritin 44, 9.6.15 2 Aldolase 158, 11.9.28 3 Conalbumin 75, 13.39.37 4 Ovalbumin 43, 14.25.42 V O Blue dextran 2,, 7.14 8
Figure S4. Calibration of the molecular weight using the calibration results of the SEC column. A: SEC chromatogram for the molecular weight markers and blue dextran eluting at the void volume. 1: Ferritin, 44 kda; 2: Aldolase, 158 kda; 3: Conalbumin, 75 kda; 4: Ovalbumin, 43 kda.; B: Standard curve for the molecular weight based on the results of SEC analysis of the molecular weight markers and blue dextran. 9
Table S2. Elution volumes and Kav for the peaks representing the cross-linked BAPs CY1 CY1 CY1 CY3 CY3 CY3 CY3 No.1 No.2 No.3 No.4 No.1 No.2 No.3 No.4 VE[mL] 9.5 1.23 11.19 13.19 9.4 1.17 11.17 13.9 Kav[ ].14.18.24.36.13.18.24.35 D e d u c e d m o le c u la rw e ig h t[ k D a ] CY1 5 A 4 3 2 1 D e d u c e d m o le c u la rw e ig h t[ k D a ] 5 1 2 3 4 1 2 3 4 B 4 3 2 1 Figure S5. The deduced molecular weights of the cross-linked BAPs. A: cross-linked CY1-BAP; B: cross-linked CY3-BAP. The numbers correspond to the peaks numbered in Figure 3 in the Results & Discussion section. 1
/kda 255 15 12 76 52 38 1 2 3 4 1 2 3 4 /kda 255 15 12 76 52 38 31 24 BSA OVA 17 12 1 2 3 4 Lysozyme 1 2 3 4 EGFP 1 2 3 4 Avidin Figure S6. Results of the co-cross-linking reactions of various proteins with Y-tagged BAPs.1: no treatment; 2: HRP treatment of each protein; 3: co-cross-linking reaction with CY1-BAP; 4: co-cross-linking reaction with CY3-BAP. The red dotted rectangles highlight the protein bands for the products of either self- or co-cross-linked products of each protein. 11
3. REFERENCE (S1) Tanaka, Y., Tsuruda, Y., Nishi, M., Kamiya, N.,and Goto, M.(27) Exploring enzymatic catalysis at a solid surface: a case study with transglutaminase-mediated protein immobilization. Org. Biomol. Chem. 5, 1764 177 12