Protein Crystallisation using Microemulsions

Abstract

X-ray crystallography is the most powerful technique for structural determination of proteins, a vital research tool, enabling insight at the atomic level, of the three dimensional structure of key protein receptors for potential drug compounds. To be successful, single, high quality crystals of the compound in question are required. Current methods to produce crystals involve frustratingly long timescales, extensive trial and error using large amounts of material, and have no guarantee of success. Polymorphic compounds add another level of frustration, requiring the thermodynamic control of crystallisation in order to overcome Ostwald’s rule of stages, which considers crystallisation from bulk solution to be under kinetic control with metastable polymorphs often crystallising initially. These factors have led to what is currently referred to as the “bottle-neck” of protein crystallisation, an acute problem motivating the rapid development of protein crystallisation techniques.
This thesis aims to alleviate the bottle-neck found in protein crystallisation by exploring protein crystallisation using microemulsions; a technique, which until now, has only been successfully applied to the thermodynamic control of crystallisation for small compounds, such as 5-methyl-2-[(2-nitrophenyl)amino]-3-thiophenecarbonitrile (commonly known as ROY) and glycine. Through the application of several different surfactant systems for the crystallisation of model protein, Lysozyme, this thesis explores the use of microemulsions with the aim of producing high quality single crystals suitable for X-ray diffraction experiments.
Numerous, large, high quality, single crystals of Lysozyme were successfully grown using a TritonX-100/1-hexanol surfactant system in which an anti-solvent, mixed microemulsion method was applied. Small angle X-ray scattering (SAXS) was effectively used to confirm formation of microemulsions and led to the determination of droplet sizes using generalised indirect Fourier Transform (GIFT) analysis. X-ray diffraction experiments showed single crystals grown from microemulsions to have a high internal order, with the resultant data sets of a publishable quality and of a comparative quality to data sets collected from crystals grown using standard vapour diffusion crystallisation techniques.
This thesis demonstrates, for the first time, that microemulsions can be successfully used to produce high quality, single crystals of the protein, Lysozyme, shining light on this novel technique as a potential means of relieving the bottle-neck of protein crystallisation. Future directions of this work include exploring the robustness of the microemulsion crystallisation technique with other proteins such as insulin, glucose isomerase and albumin. Expanding this novel technique to the crystallisation of membrane proteins may be initially explored through the crystallisation of a 25 residue, membrane spanning part of the M2 protein of the influenza virus. This would provide an interesting starting point due to the proteins’ biological importance as a target of anti-influenza drugs.