Experimental phase diagrams to optimise membrane protein crystallisation

  • My Nguyen

Student thesis: Doctoral ThesisDoctor of Philosophy (PhD)

Abstract

Membrane proteins account for approximately 30 % of all proteins and 50 – 60 % of current drug targets. The function of any membrane protein is closely related to its structure. However, obtaining the well-diffracting membrane protein crystals required for X-ray crystallography remains a major bottleneck. As a result, just over 1,300 unique membrane protein structures are known compared to more than 150,000 structures for globular proteins. In particular there is a lack of knowledge of the thermodynamic and kinetic processes underlying this process. Equilibrium phase diagrams have been widely used for globular proteins and have provide practical information on the thermodynamic requirements for crystallisation to take place. While not generally used for membrane protein crystallisation due to the complexity of measuring them, the lack of growth in the number of membrane protein structures obtained yearly by X-ray crystallography calls for more rational approaches for crystallisation.

Here, methods have been developed to determine state diagrams, as an efficient compromise to ‘true’ phase diagrams and ‘working phase diagrams’ for Outer membrane protein G (OmpG) of Escherichia coli (E. coli). These state diagrams were generated by recording all outcomes of a vapour diffusion screen combined with data reduction of the myriad of solution conditions trailed in the screen to scale the net effective attractive interactions in the system. Identifying the nucleation zone from the state diagram allowed for scaling-up of crystallisation experiments. This has led to an optimised crystallisation protocol for OmpG, the insights from which were then applied to the crystallisation of the Outer membrane protein X (OmpX). Optimisation of OmpG crystallisation resulted in obtaining a novel crystal structure for OmpG in the space group P 43 21 2 with crystal sizes up to 880 μm at 2.7 Å resolution.

Furthermore, the kinetic pathways leading to crystallisation were also explored, which revealed a very complex interplay between protein and detergent phase behaviour, which was necessary to produce protein crystals.
Date of Award22 Mar 2022
Original languageEnglish
Awarding Institution
  • University of Bristol
SupervisorJennifer McManus (Supervisor)

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