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It was important to make sure that conditions were compatible for both BPTI and PDI and that the two proteins did not aggregate after mixing. This possibility was already minimised by testing BPTI constructs in an NMR buffer previously used for NMR with PDI constructs.

12 11 10 9 8 7 6 5 4 3 2 1 kDa 188 98 62 49 38 28 17 6 14 PDI BPTI Mixed BPTI/PDI Samples 12 11 10 9 8 7 6 5 4 3 2 1 kDa 188 98 62 49 38 28 17 6 14 PDI BPTI Mixed BPTI/PDI Samples

Figure 5.4: SDS-PAGE of (30-51, 5-14) BPTI, PDI and (30-51, 5-14) BPTI/PDI combined samples. Lane 1, SeeBlue marker; lanes 2-4, (30-51, 5-14) BPTI samples; lane 6, (30-51, 5- 14) BPTI:PDI 5:1 (30μM BPTI, 6μM PDI); lane 7, (30-51, 5-14) BPTI:PDI 1:1 (30μM each), lane 9, PDI ion exchange elute; lane 10, flow through from buffer exchange of PDI sample; lane 11, PDI after buffer exchange to 10 mM ammonium bicarbonate; lane 12, PDI after lyophilisation and resuspension in NMR buffer.

Figure 5.4 shows SDS-PAGE analysis of (30-51, 5-14) BPTI intermediate, PDI and a mixture of samples at different molar concentrations. It clearly shows monomeric PDI which can be purified, lyophilised and resuspended in NMR buffer without dimerisation or degradation occurring (lanes 9-12). As previously shown, BPTI samples were also prepared successfully in NMR buffer (lanes 2-

molecular mass than BPTI (6.6 kDa), therefore will show up as a much larger band when at the same molar concentration (lane 7). Only at a 5 times higher molar concentration of BPTI does the PDI band appear a similar size (lane 6).

5.4. (30-51, 5-14) BPTI interaction with PDI

Since a partly-folded protein would be more likely to interact with PDI than one that is natively folded, it seemed logical to begin interaction studies using (30-51, 5-14) BPTI.

Before the addition of PDI, an HSQC spectrum was acquired with (30-51, 5-14) BPTI only. Since no prior knowledge was available with regard to the binding of PDI to a partly folded protein, initially the enzyme was added at a 1:1 molar ratio of BPTI:PDI. However, at this ratio it was found that very little was detected in the HSQC spectrum (data not shown). With a second sample, a sub- stoichiometric amount of PDI was added, at a BPTI:PDI ratio of 5:1. Surprisingly, even at this low sub-stoichiometric level of enzyme only a few peaks were visible in the HSQC spectrum, Figure 5.5. Overlay of the (30-51, 5-14) BPTI HSQC spectrum without PDI enabled the remaining peaks to be identified. Two clear peaks are visible for Ala58, with Gly57 and Gly36 also visible.

Figure 5.5: HSQC spectrum of (30-51, 5-14) BPTI in the presence of PDI at a BPTI:PDI ratio of 5:1.

Since BPTI is a relatively small protein (6.6 kDa), it is well suited to investigation using various NMR techniques, since such a small protein will have relatively fast molecular tumbling, enabling sharp peaks to be detected even at low temperatures. By comparison, PDI is a relatively large protein (56 kDa), resulting in a much slower molecular tumbling rate, making it too large to be detected using standard NMR techniques. When combined with 15_{N labelled}

BPTI, the BPTI/PDI complex reaches 62.6 kDa, so is too large to be detected in the HSQC spectrum. So, generally speaking, peaks in the HSQC spectrum using15N labelled BPTI will only be visible for the unbound protein, but not when bound to the PDI enzyme. However, even in the bound state, regions of BPTI that remain flexible may still be observed, since they will still be able to show fast motions locally.

molecules have been bound by PDI, despite the enzyme being present at only one fifth of the molar concentration.

Theoretically, this could be explained if PDI had multiple binding sites. However, previous studies suggest that PDI only has a single binding site (Pirneskoski, Klappa et al. 2004). Rather, it seems that PDI is able to bind and unbind rapidly from its partly-folded substrate, such that all (30-51, 5-14) BPTI will have been bound within the NMR timescale. The drastic loss of signal even with PDI at one fifth of the molar concentration of BPTI may suggest that the enzyme is binding to at least 5 substrate molecules within the timescale of HSQC observation. Alternatively, binding may be less rapid, but exchange between bound and unbound BPTI may be on an intermediate timescale, resulting in some line broadening even in the unbound state.

Interestingly, however, a few peaks remain visible in the HSQC spectrum of Figure 5.5. This could simply be because these residues had a higher intensity in the unbound BPTI intermediate, and so when only a small fraction of the population remains in the unbound state, only these residues remain detected. Gly57 and Ala58 in particular showed relatively high peak heights in the HSQC spectrum of unbound (30-51, 5-14) BPTI at 5°C (see Appendix C).

Alternatively, those residues that remain visible may be able to experience some “local tumbling”, independent of the rest of the molecule. Perhaps the flexibility of the C-terminal region allows it to retain isotropy by moving unhindered by the rest of the molecule, thus permitting detection of HSQC signal from this region.

To get a better idea of the extent of PDI binding, HSQCs of15N labelled (30-51, 5-14) BPTI were obtained again, but this time titrating in smaller quantities of PDI enzyme. The enzyme was added at BPTI:PDI ratios of 200:1, 100:1, 50:1, 25:1, 10:1 and 5:1.

A

Figure 5.6: HSQC spectra of (30-51, 5-14) BPTI interaction with PDI. A) (30-51, 5-14) BPTI only; B) 50:1 ratio BPTI:PDI; C) 10:1 ratio BPTI:PDI; D) Overlay of spectra from A, B and C. Assigned peaks from (30-51, 5-14) BPTI only spectrum are labelled.

Comparison of each spectrum with a (30-51, 5-14) BPTI only control (no enzyme) showed very little difference at the 200:1 ratio (not shown). At 100:1

C

ratio, all assigned peaks are still present, but a reduction in intensity is clear. At 50:1, a further reduction of peak intensity is clear, with some assigned peaks no longer visible (Figure 5.6B). This trend continues as the molar concentration of PDI is increased. At 10:1 ratio, most assigned peaks are no longer visible, and most that remain are just above the noise level (Figure 5.6C). An overlay of HSQC spectra with various quantities of PDI illustrates the peaks that disappear most rapidly and those that persist even at higher concentrations (Figure 5.6D).