(NH/ 15 N) (ppm) Peptide II Peptide I Peptide III Peptide IV Peptide V 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 15 N 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

N3 5 Q57 V5 0 N4 4 N2 1 Q6 4 N2 8 K4 5 N2 8 S1 9 S2 5 D4 9 E5 2 E4 2 N2 1 N4 4 Q4 0 E2 9 E6 8 Q5 5 K5 6 * Q4 0 L2 7 S3 2 K6 9 H5 3 A3 9 Q6 4 Q5 7 A6 3 V1 7 A2 0 * K2 6 L6 1 A6 7 A2 3 V1 1 A6 0 K7 0 L5 4 R1 6 V7 1 K3 0 R3 7 K1 8 I1 0 * R4 1 I2 4 H1 3 N3 5 K6 5 D5 9 D1 4 V6 2 Q5 5 M 48 E3 4 D6 6 D2 2 S5 M3M3 L3 8 I3 3 E1 5 L5 8 R3 6 A2 E7 I6 A9 R1 2 V4 3 K8 V4 K5 1 A4 6 D Figure 4.7 Assigned Sbi IV HSQC 1 H- 15 N HSQC spectrum of uniformly 15 N-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% D 2 O, 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.

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 15 N 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. * Sbi IV residue number (NH/ 15 N) (ppm) 99 * * * * * * * *

Sbi IV residue number Peak height decrease (%) 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%. 101

Appendix 5: Efb NMR table of assignments Column 1- construct numbering; column 2 – Efb numbering 161

Appendix 5 continued 162

Appendix 6: Sbi IV NMR table of assignments Column 1- construct numbering; column 2 – Sbi IV numbering 163

Appendix 6 continued 164