(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