Protein crystallisation using low-cost electric-field assisted setups for microbatch, vapour diffusion, and in situ diffraction studies

Abstract

Understanding the molecular structures of proteins is fundamental to elucidating their molecular roles and mechanisms within living organisms. Achieving high-quality protein crystals is essential for successful X-ray crystallography, enabling the determination of protein structures. The efficacy of crystallisation methods directly impacts the physical, chemical, and biological properties of the resulting crystals, highlighting the critical need for precise control over the crystallisation process outcomes. In this thesis, four experimental setups were designed and fabricated using CAD design and 3D printing. These setups allowed the investigation of the effects of electric fields on protein crystallisation employing techniques such as microbatch under oil and vapour diffusion. The influence of the electric field on various aspects of protein crystallisation, including nucleation rates, crystal size, and quality, was analysed using Hen Egg White Lysozyme (HEWL) and previously uncharacterised RNA Editing Mediator Complex 1 (REMC1). The experimental results demonstrated enhanced HEWL nucleation, producing a higher number of crystals with smaller sizes and narrower size distribution when the electric field was applied. This effect was interpreted by modifying the chemical potentials and the diffusion rates in the crystallisation process by the electric field. In addition, HEWL crystals were subjected to in situ X-ray diffraction studies, revealing an enhancement of the internal order of the crystals by electric field. This was evident by the lower mosaic spread and Wilson B-factor, which is attributed to the alignment of molecular dipoles by electric fields, resulting in an enhanced internal lattice order. In contrast, the quality of the REMC1 crystals was not improved noticeably by applying the electric field. This was likely due to the poor intrinsic quality of the crystals. The findings underscore the potential for electric fields to optimise crystallisation processes, offering significant implications for both theoretical understanding and practical applications in fields such as structural biology and pharmaceutical development. The thesis contributes to a deeper understanding of the crystallisation process and opens avenues for advancements in drug design and structural biology research.