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Peptide I. (NH/ 15 N) (ppm). Peptide II. (NH/ 15 N) (ppm). Peptide III. (NH/ 15 N) (ppm). Peptide IV. (NH/ 15 N) (ppm). Peptide V. (NH/ 15 N) (ppm). Efb residues A29 – R165. Figure 3.10 Chemical shift perturbation of Efb upon titration of fibrinogen peptides
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Peptide I (NH/15N) (ppm) Peptide II (NH/15N) (ppm) Peptide III (NH/15N) (ppm) Peptide IV (NH/15N) (ppm) Peptide V (NH/15N) (ppm) Efb residues A29 – R165 Figure 3.10 Chemical shift perturbation of Efb upon titration of fibrinogen peptides The x-axis represents Efb residues A29 – R165. The combined NH proton and 15N chemical shift change is represented by the y-axis (blue bars). The combined chemical shift perturbation for each residue of Efb is shown for each peptide. In each case, the Efb:peptide molar ration was 1:1. Where data are absent, the peak either could not be assigned in the starting spectrum or, if it was, it could not be tracked throughout the titration. The largest perturbations are seen for I78, Q134 and L159 in the presence of peptide V. 53
N35 Q57 V50 N44 N21 Q64 S19 N28 N28 S25 D49 Q40 E42 E52 N44 K45 E29 Q55 N21 E68 * L27 K56 D59 K65 A39 Q40 H13 H53 Q64 K69 D14 V62 N35 R16 M48 M3 L54 Q57 R41 S32 S5 Q55 V71 * D66 V17 A63 E34 K18 L38 I24 84 R37 I10 D22 K30 A60 V43 A20 K70 R12 K8 * L61 V4 K26 I33 L58 A67 E15 A2 I6 A23 K51 V11 R36 A9 E7 A46 D31 Figure 4.7 Assigned Sbi IV HSQC 1H-15N HSQC spectrum of uniformly 15N-enriched Sbi IV recorded at 16 ºC and 600 MHz. The sample contained 5 mM MES, 100 mM sodium chloride, 1 mM EDTA,1 mM benzamidine and 10% D2O, pH 5.5. The concentration of Sbi IV in the samples as determined by UV spectroscopy was ~1 mM (8.33 mg ml-1). Peaks are labelled with the residue number for the construct. Peaks A2 and M3 are derived from the vector. Peaks V4 - V71 correspond to residues V198 – V265 of Sbi IV. Pairs of peaks from the side chain amides of asparagine and glutamine are connected with horizontal lines. For the table of assignments refer to appendix 6.
* (NH/15N) (ppm) * * * * * * * * 99 Sbi IV residue number Figure 5.6 Chemical shift perturbation of Sbi IV upon C3d binding Sbi IV residue numbers are shown on the x-axis. The combined NH proton and 15N chemical shift change is represented by the y-axis (blue bars). Where peaks split (orange bars), the average change across all peaks is shown. Where peaks broaden beyond detection, the chemical shift perturbation which occurred up to the point of disappearance is shown. Peaks that broaden beyond detection are marked with asterisks. No data are shown for K224 or V211 as these peaks could not be tracked throughout the titration experiment. Data does not exist for P241.
Peak height decrease (%) 101 Sbi IV residue number Figure 5.8 Sbi IV peak height decrease upon C3d binding Sbi IV residue numbers are shown on the x-axis. The decrease in peak height is represented by the y-axis (blue bars). Where peaks split (orange bars), the average decrease across all peaks is shown. Where peaks broaden beyond detection, the percentage decrease is 100%.
Appendix 5: Efb NMR table of assignments Column 1- construct numbering; column 2 – Efb numbering 161
Appendix 6: Sbi IV NMR table of assignments Column 1- construct numbering; column 2 – Sbi IV numbering 163
