Purification of E5

A peptide corresponding to the TM domain of E5:

KKKFLGLVAAMQLLLLLFLLLFFLVYWDHK

was ordered from a commercial manufacturer, and provided in a crude form con- taining truncated products and protecting groups from the solid-phase synthesis. It was purified using reverse-phase HPLC (RP- HPLC) as detailed in section 2.6.3 and then analysed by mass spectrometry.

Elution fractions corresponding to major peaks in the HPLC chromatogram (Fig- ure 5.23) were collected and pooled. The peak at around 46.3 minutes (marked with (*)) was found to contain E5. This was deduced from the mass spectrometry data (Figures 5.24a & 5.24b) which revealed a peak at 3662.2 Da in the deconvoluted spectrum, which is in good agreement with the theoretical mass of 3664.60 Da.

Figure 5.23: HPLC chromatogram for the purification of E5

The pure E5 peptide was stored in TFE. The concentration was measured using ab- sorbance at 280 nm and Beer-Lambert Law. E5 was then titrated into the PDGF R sample by first drying it under a stream on nitrogen such that a thin film was formed. This film was then dissolved in a solution containing PDGF R-TM-20 in DPC/DPPC mixed micelles.

(a) Mass spectrum of E5

(b) Deconvoluted mass spectrum of E5

Figure 5.24: Assessment of purified E5 through ESI-MS. Pooled fractions from the HPLC purification were checked for the existence of the peptide and it’s purity. The top panel (a) shows the mass spectrum with multiple charge states of the protein.

5.6

Summary and Conclusion

This chapter began by detailing the modifications that were carried out to purify PDGF R-TM-20 which was expressed using an existing plasmid construct with an (rEK) enterokinase cleavage site. This was important because even though a protocol existed in the Dixon lab for the purification of unlabelled PDGF R-TM-20, it needed to be ensured that yields were maximised when the protein was expressed in expensive labelled media. The changes involved switching the chaotropic agent to urea, changing the volumes of elution and wash bu↵ers and the concentration of imidazole in them. Also, the choice of detergent was changed to Triton X-100 as it could easily be removed using Bio-Beads for reconstitution into lipid bilayers. It was subsequently observed that despite optimising the cleavage conditions, it would not be economically feasible to use rEK for cleavage for the quantities of protein required for NMR. Additionally, the construct contained an uncleavable C- terminal His-tag whose removal would require genetic manipulation. In light of these difficulties, a new construct was created using TOPO cloning with a His-tag cleavable using TEV protease. The construct was created exactly as per requirements and TEV protease was chosen because protocols existed in the lab to produce it in large quantities in an economically efficient manner.

However, it soon became apparent that the self-cleaving MBP which is produced when expressing TEV protease cannot be removed from the sample using any of the purification methods that were tried (section 5.3.4). A GFP-tagged TEV protease was then acquired which resolved this issue and samples could finally be made for NMR analyses.

A protocol for the reconstitution of PDGF R-TM-20 into liposomes using Bio-Beads was adopted which monitored the removal of detergent using solution-state NMR. Insertion and secondary structure classification was studied using CD which revealed a highly helical structure of the protein within Triton X-100 detergent and POPC liposomes.

Two-dimensional13C–13C DARR correlation spectra were acquired of the liposomes after inserting PDGF R-TM-20 labelled with [1-13C]glucose. Peaks were observed in the aliphatic as well as carbonyl regions of the spectrum. However, not only was the signal to noise ratio very low, but the number of peaks were significantly lower than expected such that meaningful distance restraints could not be obtained. This necessitated a re-examination of several aspects of the sample preparation process. It may be that instead of spontaneously inserted into the membrane upon detergent removal, a certain percentage of the protein is aggregating. Approaches to circumvent this could involve changing the bilayer composition, or the length of the juxtamembrane region. A more traditional uniform labelling scheme could also be adopted. Due to time-constraints, this problem could not be immediately addressed.

The last section of this chapter dealt with the sample preparation methods for the investigation of E5/PDGF R-TM interactions using solution-state NMR. A dou- ble purification method involving IMAC and HPLC were adopted to obtain pure PDGF R-TM-20 for analyses. The following chapter will address in detail the solution-state NMR experimental optimisations performed and the results obtained from these studies.

A Solution-State NMR study of

E5/PDGF R Transmembrane

Interactions

6.1

Introduction

Solid-state NMR is a very powerful technique that can be employed to investigate structural properties and interactions in a lipid bilayer, however, current data ac- quisition methods render it far less sensitive than its solution counterpart. Where cutting-edge methods are being developed for structural assignments of proteins in ssNMR, assignment methods using 3D NMR are well-established in the solution state. In light of this, it was decided to investigate E5/PDGF R interactions using solution-state NMR as a complementary method.

Solution-state NMR requires fast tumbling on the NMR timescale in order to average anisotropic NMR interactions (e.g. CSA), therefore large biomolecules or complexes > 50 kDa pose significant difficulty in yielding adequate resolution and signal to noise ratio. As such, liposomes do not constitute a suitable membrane-mimetic for studying membrane embedded proteins using solution-state NMR. Detergents, on the other hand, have been used to study membrane proteins in vitro for the past 40 years (Helenius and Simons, 1975; Tanford and Reynolds, 1976) and most NMR structures of membrane proteins have been solved in detergent solutions (Kang and Li, 2011; Warschawski, 2013).

This chapter will introduce NMR experiments performed on uniformly15N labelled PDGF R-TM-20 in detergent micelles and establish a suitable system for acquiring more complex 3D experiments. A set of resonances was hypothesised to belong to the TM region of PDGF R-TM-20 through Hydrogen–deuterium exchange experiments. PDGF R-TM-20/E5 interactions were explored by titrating E5 into the PDGF R- TM-20 mixed micelle solution and the implications of the results obtained will be described.