Characterising Disordered Proteins of the Cancer Genome using Biophysical Techniques

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Abstract

Protein function and dysfunction, and their intimate ties to protein structure, has been a core focus of research for several decades. More recently, research into the lack of structure in proteins has reached fever pitch. Intrinsically disordered proteins (IDPs) are proteins (or protein regions) that exist as collapsed or extended, dynamically mobile conformational ensembles, either at secondary or tertiary level, whilst remaining biologically active. The properties of IDPs can impede their study; they are often inherently unstable, are vastly wide-ranging in molecular weight and often difficult to express in large quantities. Mass spectrometry (MS) has evolved into a tool for the study of dynamic systems such as IDPs due to its large dynamic range, high sensitivity, low sample consumption and its lack of bias towards the folded state of a protein. The addition of ion mobility separation to mass spectrometry analysis (IM-MS) provides insight into the conformations adopted by proteins and their complexes, measuring their rotationally averaged collision cross section which can be compared with coordinates from other biophysical techniques such as X-ray crystallography, NMR and to molecular modelling. The work presented in this thesis uses both MS and IM-MS, along with several other biophysical techniques, to interrogate a number of IDPs which are implicated in cancer.Firstly, variable temperature IM-MS is used to probe several proteins of increasing disorder; structured protein cytochrome c, the tumour suppressor protein p53 and the oncoprotein Murine Double Minute 2 (Mdm2), performing IM-MS measurements at a range of temperature from 200 K to 571 K to elucidate the gas-phase unfolding behaviour of each protein. The interaction between p53 and Mdm2 is a current target for cancer drug therapy. Hence MS and IM-MS, alongside circular dichroism and hydrogen-deuterium exchange are next employed to determine the effect of several known small molecule ligands on the conformations adopted by these disordered domains. The significant structuring of both of these disordered proteins upon binding to their respective ligands can be observed using IM-MS, but is not apparent when using other biophysical techniques, highlighting the ability of IM-MS to capture conformational changes occurring in solution on a short timescale. The regulation of disorder in cells is postulated to be mediated by proline residues. I investigate the impact of proline replacement on the populations of conformers presented by p53 using a range of mutants and then go on to study how these mutations impact upon the binding stoichiometry, affinity and conformational preference of p53 for its interaction partner Mdm2. Finally, the disordered melanoma associated antigen 4 MAGE-A4, and its ability to bind to p53 and block its transcriptional activity is probed using MS and IM-MS.

Keyword(s)

IDP; Protein; mass spectrometry; p53

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