Carmichael, Joshua; (2024) Structural and Biophysical Analysis of Factor H Related Protein 5. Doctoral thesis (Ph.D), UCL (University College London).

Text Joshua Carmichael PhD Thesis - CORRECTED.pdf - Accepted Version Access restricted to UCL open access staff until 1 June 2025. Download (8MB)

Abstract

Factor H related protein 5 (FHR5) is an element of the alternative pathway of complement and a member of a subset of proteins within this system termed the Factor-H related proteins. Originally discovered in complement deposits in the glomerulus, it holds particular medical significance due to its role in a form of C3 glomerulopathy termed CFHR5 nephropathy, which is an important cause of renal failure in Cyprus. However, FHR5 in the context of human physiology remains largely uncharacterised, with the notable exception of a Small Angle X-ray Scattering (SAXS) derived solution structure. To investigate the structure and function of FHR5, and to a lesser extent FHR1 and FHR2, a variety of different complement proteins were purified. These were explored by structural techniques (x-ray crystallography, SAXS, analytical ultracentrifugation) and techniques for measuring protein-protein interactions (surface plasmon resonance and biolayer interferometry). X-ray crystallography was used to characterise the dimerization domain of FHR5 to a resolution of 2.3Å, showing a homodimer stabilised by hydrophobic interactions, forming a snugly fitting, antiparallel structure. Previous SAXS results for FHR5 were also reproduced. FHR2 was characterised by SAXS, revealing a radius of gyration of 45Å and a maximum dimension of 144Å. Crystallisation trials for both FHR5 and FHR2 were also undertaken, using the technique of Microseed Matrix Screening (MMS). I have sought to test the hypothesis that FHR5 is preferentially recruited to surfaces with a high surface density of C3 breakdown fragments, displaying avidity once molecules of C3b or C3d are within steric range of both of its binding sites. To do this, surface plasmon resonance (SPR) and Biolayer Interferometry (BLI) were used. SPR was also employed to test for evidence of competition between Factor H and FHR5 for C3b.

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