Developing novel methods for co-translational studies of membrane protein folding

Abstract

Membrane proteins are a diverse range of macromolecules and when misfolded, disease phenotypes often occur. Membrane protein folding studies ascertain how a linear polypeptide forms its final functional three-dimensional structure. Current methods for studying membrane protein folding rely on sophisticated biophysical methods to probe the folded state of a purified protein in detergent micelles or an artificial lipid mimetic. However, most proteins in vivo fold in a co-translational process, from N- to C- terminal as the peptide emerges from the ribosome. This thesis develops new methods to expand the toolbox of co-translational folding studies for membrane proteins. Firstly, I develop a new method for purification of a 6 TM Rhomboid protease in a partially translated state while still attached to the ribosome to form a ribosome-bound nascent chain complex (RNC). This RNC purification method utilises a novel polymer-based system to not only capture the partially expressed protein, but also its surrounding lipids from its native E. coli membrane allowing a more physiological snapshot of the co-translational folding process, without the need to first purify using detergents as is common in the field. I also show evidence that the SecYEG bacterial translocon can co-purify with the RNC complex which opens many avenues of structural and biochemical research projects, of which these RNC samples are highly amenable to. The final chapter of this thesis focuses on the development of an atomic-force microscopy (AFM)-based protein unfolding experiment for a fully translated and reconstituted membrane protein. This approach can be adapted to further study the effects of lipid environment, and intrinsic stability of membrane protein RNCs at varying positions through their translation cycle which should revolutionise methods for studying co-translational membrane protein folding.

Date of Award	1 Mar 2021
Original language	English
Awarding Institution	King's College London
Supervisor	Paula Booth (Supervisor)