Development of nuclear magnetic resonance methods for determination of membrane protein structure

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Abstract

Membrane proteins represent over a third of all proteins encoded for by the human
genome and play a vital role in the functionality of the cell, by controlling a vast number of
cellular processes. With over half of pharmacological drugs targeting membrane proteins,
their importance is not to be under estimated. Yet the number of three-dimensional
membrane protein structures reported to date falls well short of that of their water soluble
counterparts. This discrepancy can directly be attributed to the difficulties involved in
studying membrane protein structure due to their hydrophobic nature, resulting in a
number of challenges in the production and purification of protein, whilst requiring the use
of a suitable membrane mimetic upon extraction from their native membrane.
Solid state NMR (ssNMR) as a technique for studying membrane protein structure is well
placed in being able to obtain structural information for membrane proteins in “native-like”
lamellar bilayer environments but there are challenges involved in preparing suitable
samples for analysis. As there is no “one suit fits all” method for preparing membrane
protein samples for ssNMR analysis, conditions that result in fully reconstituted protein, that
also allow for high resolution structural analysis have to be trialled.
This study presents work on sample preparation methods for the reconstitution of the small
alpha helical transmembrane (TM) proteins, using the well characterised TM protein
Glycophorin A (GpA) as a model peptide. Established biophysical and NMR techniques were
used to characterise DMPC lipid embedded peptides prepared using two reconstitution
techniques. The limited site specific labelling at key positions of the GpA homodimer was
used to evaluate the feasibility of using similar sample preparation and labelling schemes
when applied to that of the Bovine Papillomavirus E5 (BPV E5) TM protein, for which no
solved three-dimensional structure exists. Characterisation of the DMPC membranes into
which membrane proteins where reconstituted was also conducted. To compliment ssNMR
analysis of BPV E5, preliminary work on the use of fast tumbling isotropic bicelles to study
membrane protein structure by solution NMR is also presented.

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