(NH/15N) (ppm)

(NH/15N) (ppm)

(NH/15N) (ppm)

(NH/15N) (ppm)

(NH/15N) (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 peptidesThe 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

Q57V50 N4

4 N21Q64

N28

K45

N28

S19

S25

D49 E5

2

E42

N21

N44

Q40

E29

E68

Q55

K56

*

Q40

L27

S32

K69

H53

A39Q64Q57

A63

V17

A20*

K26

L61 A6

7A23V1

1

A60K70

L54

R16V71

K30

R37

K18 I1

0

*R41I24

H13N35

K65

D59

D14

V62

Q55

M48

E34

D66

D22

S5M3

L38

I33 E1

5

L58

R36

A2

E7

I6

A9

R12

V43

K8

V4

K51A46

D31

84

Figure 4.7 Assigned Sbi IV HSQC1H-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.

Figure 5.6 Chemical shift perturbation of Sbi IV upon C3d bindingSbi 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.

*

Sbi IV residue number

(NH/15N)

(ppm)

99 *

*

*

*

**

*

*

Sbi IV residue number

Peak height decrease

(%)

Figure 5.8 Sbi IV peak height decrease upon C3d bindingSbi 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

App

endix 6: Sb

i IV N

MR

table of assignm

ents

Colum

n 1- construct numbering; colum

n 2 – Sbi IV

numbering

163

Appendix 6 continued

164