Nash, Anthony (Researcher in chemistry) (2014) Understanding the "rules of engagement" for membrane protein folding : chemical biology and computational approaches for determination of structure and dynamics. PhD thesis, University of Warwick.

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Abstract

Approximately one third of genes in the human genome (1) encode transmembrane (TM) proteins and form more than half of all drug targets (2). However, our understanding of how these proteins fold into their functional form, as well as how they may misfold into a disease-associated form, remains a difficult area of study. By observing the effects of single point mutations in the context of a native sequence, in addition to adding and mutating interhelical interaction motifs on a low complexity sequence background, we aim to elicit ‘rules’ of TM protein domain association.

For the single point mutation in the context of a native sequence, the TM domain of the sequence Neu, along with its oncogenic substitution V664E form Neu*, were selected. Using molecular dynamics (MD) a united atom model of each dimer in a model bilayer system was subjected to umbrella sampling along an interhelical reaction coordinate to yield a free energy profile of self-association. The lipid order, bilayer thickness, and peptide tilt angle were calculated from trajectories taken from three points along the reaction coordinate. Helical composition, solvent accessible surface area, and hydrogen bond analysis (for the V664E substitution) were performed at the free energy minimum.

Low complexity sequences of polyleucine and polyleucine-alanine heptad repeat sequences, with and without interaction motifs similar to those present in the Neu model, were ligated into PBLM100 plasmids. Transformed E. coli cells were subjected to semi-quantitative homo-interaction analysis using the GALLEX assay. The same TM sequences were modelled using a coarse grained (CG) forcefield. Umbrella sampling along an interhelical reaction coordinate was performed to yield a free energy profile of self-association. Single-linkage cluster analysis of peptides was performed at the global free energy minimum. A representative structure from each set was compared to an averaged structure from the clusters of an atomistic conformational search.

The results presented in this study, could contribute to what in theory would be a large database of motif-driven rules for TM helical domain oligomerisation. This may encourage further investigation into TM protein design for novel application.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QP Physiology
Library of Congress Subject Headings (LCSH):	Membrane proteins, Protein folding
Official Date:	February 2014
Dates:	Date Event February 2014 Submitted
Institution:	University of Warwick
Theses Department:	Molecular Organisation and Assembly in Cells
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Notman, Rebecca ; Dixon, Ann M.
Sponsors:	Engineering and Physical Sciences Research Council
Extent:	xviii, 248 leaves : illustrations, charts
Language:	eng

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