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Title: Biophysical studies of protein assemblies
Author: Wicky, Basile Isidore Martin
ISNI:       0000 0004 7653 8915
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2019
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Proteins are synthesised as linear polymeric chains. The subtle energetic interplay of interatomic interactions results in chain folding, through which proteins may acquire defined structures. This spatial organisation is encoded by the protein sequence itself; the so-called thermodynamic hypothesis formulated by Anfinsen in 1961. A defined structure is often considered a pre-requisite to protein function, but widespread existence of intrinsically disordered proteins (IDPs) has prompted a re- evaluation of the ways biological function may be encoded into polypeptide chains. Furthermore, proteins often exist as part of multi-component entities, where regulation of assembly is integral to their properties. The interplay between disorder, oligomerisation and function is the focus of this thesis. Some IDPs fold conditionally upon interacting with a partner protein; a process known as coupled folding and binding. What are the biophysical advantages and consequences of disorder in the context of these interactions? A common feature of IDPs is their sequence composition bias, with charged residues being often over-represented. It is therefore tempting to speculate that electrostatic interactions may play a major role in coupled folding and binding reactions. Surprisingly, the opposite was found to be true. Charge-charge interactions only contributed about an order of magnitude to the association rate constants of two contrasting model systems. The lack of pre-formed binding interfaces-a consequence of disorder-might preclude electrostatic acceleration from complementary patches. By looking at the role of the sequence, many studies have taken a protein-centric approach to understanding disorder. Yet there is paucity of data about the effect of extrinsic factors on interactions involving disordered partners. Investigating the role of co-solutes, it was discovered that the kinetic and thermodynamic profiles of coupled folding and binding reactions were sensitive to ion-types. This effect followed the Hofmeister series, and occurred at physiological concentrations of salt. The sensitivity of coupled folding and binding reactions-a consequence of the lack of stability of IDPs-might be advantageous. Given the role of ions in biology, this 'biophysical sensing' could be a mechanism of physiological relevance, allowing modulation of protein-protein interactions involving disordered partners in response to changes in their environments. In cells, signalling networks are often multi-layered, and involve competing protein-protein interactions. The interplay between the biophysical characteristics of the components, and the behaviour of the network were investigated in a model tripartite system composed of folded and disordered proteins. The BCL-2 family regulates the intrinsic pathway of apoptosis through control of mitochondrial outer-membrane permeabilisation; a result of BAK and BAX oligomerisation. Through a shared homology motif (termed BH3), the subtle balance of their interactions determines cellular fate at the molecular level. Characterisation of the model under simple biochemical conditions revealed large differences in affinities among binary interactions; the consequence of the lifetime of the complexes, not their speed of association. A membrane-like environment, re-created using detergents, allows the oligomerisation of BAK and BAX in vitro. Furthermore, investigation of the tripartite system under detergent conditions showed that regulation of the network was the result of competing hetero- and homo-oligomerisation events. Relationships to their biophysical properties were gained by probing their energy landscapes using protein folding techniques. The connection between the biophysical properties of the components of the network and their interactions provides a molecular explanation for the regulation of apoptosis. This thesis offers insights into the ways structured assemblies and environmentally responsive disorder elements may encode functions into proteins.
Supervisor: Clarke, Jane Sponsor: Cambridge Trust ; Wellcome Trust ; Cambridge Philosophical Society ; St Catharine's College
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Proteins ; Protein-Protein Interactions ; Intrinsically Disordered Proteins ; Molecular Biophysics ; Protein Oligomerisation ; BCL-2 ; Apoptosis ; Native MS ; Kinetics ; Thermodynamics ; Protein Folding ; Coupled Folding and Binding ; Molecular Biology ; Chemical Physics