Biophysical and computational characterisation of the disorder-to-order structural transition of the small hydrophilic endoplasmic-reticulum associated protein, SHERP

Drew, Elliot Dudley (2018) Biophysical and computational characterisation of the disorder-to-order structural transition of the small hydrophilic endoplasmic-reticulum associated protein, SHERP. Doctoral thesis, Birkbeck, University of London.

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Abstract

This thesis explores the disorder-to-order structural transition of the small hydrophilic endoplasmic reticulum associated protein (SHERP). SHERP has been shown to be essential to the life cycle of Leishmania major, a parasite responsible for leishmaniasis which kills tens of thousands every year. The protein is almost entirely disordered in solution, but undergoes a dramatic increase in helicity upon binding to anionic lipids or detergents. Although the ordered structure of SHERP had previously been solved by solution nuclear magnetic resonance spectroscopy in the presence of sodium dodecyl sulphate (SDS), both the nature of the disordered ensemble of the protein and the organisation of the SHERP/detergent complex were unknown. Using a combination of synchrotron radiation circular dichroism spectroscopy (SRCD), small angle X-ray scattering (SAXS) and molecular dynamics (MD), several projects were carried out exploring the disorder-to-order structural transition of SHERP in the presence of SDS. The effectiveness of sequence-based predictors to estimate the effect of single mutants was explored, with a number of mutants expressed and characterised by SRCD and MD. A mutant, the “permutant”, was designed with the aim of decreasing the disorder of the protein in solution while maintaining amino acid composition, by introduction of multiple potential i → i4 salt bridges created by permutations of the wild-type sequence. Molecular dynamics simulations of the wild-type and “permutant” construct found a dramatic increase in salt bridge formation, and in vitro characterisation of the “permutant” construct showed it had significantly greater helical character than the wild-type in the absence of SDS. The disordered ensemble of SHERP was characterised by replica exchange MD, SRCD and SAXS. Good agreement was found between simulation and experiment, with a predominantly unfolded ensemble deficient in secondary structure described by our results. The changes that occur upon SHERP binding to SDS were also characterised. MD simulation of the SHERP-SDS complex showed that the protein bound among the head-groups of the SDS micelle, and the helical content and helix-turn-helix structure was retained. It also allowed identification of several cationic side-chains which formed stabilising salt bridges with the sulphates of SDS. The complex was then characterised in vitro, by SAXS and CD spectroscopy. The addition of the protein led to a doubling in micelle length, with multiple SHERP molecules found to bind to the anionic head-groups in the shell of the micelle. The residues identified during the MD simulation were substituted with alanine to make a series of mutants with increasing negative charge. Significant decreases in helicity, micelle length and the numbers of protein bound occurred as negative charge increased, possibly caused by decreased affinity of the protein for the micelle causing less protein molecules to bind per micelle, leading to a decreased chance of stabilising protein-protein interactions resulting in partial folding of the protein. These results demonstrate the importance of charge-charge interactions in the disorder-to-order structural transition of SHERP, and provide structural context for future functional work on this protein.

Item Type: Thesis (Doctoral)
Copyright Holders: The copyright of this thesis rests with the author, who asserts his/her right to be known as such according to the Copyright Designs and Patents Act 1988. No dealing with the thesis contrary to the copyright or moral rights of the author is permitted.
School/Department: School of Science > Biological Sciences
Depositing User: ORBIT Editor
Date Deposited: 03 Jan 2019 13:26
Last Modified: 03 Jan 2019 13:26
URI: http://bbktheses.da.ulcc.ac.uk/id/eprint/366

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