Study of complement regulatory factor H based on Forster resonance energy transfer and investigation of disease-linked genetic variants
Pechtl, Isabell C.
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The plasma protein complement factor H (fH, 155 kDa) regulates the activity of the alternative pathway of complement activation. Factor H is monomeric, and its 20 CCP modules are arranged in a predominantly elongated conformation, joined by linking sequences that vary in length, with the longest linkers occurring in the central portion of the molecule. CCP modules 1 through 4 of fH host its capacity to act as a cofactor for fI-mediated proteolytic degradation of C3b and its ability to accelerate the decay of the C3 convertase, C3bBb, thereby regulating the so-called tick-over activation of the alternative pathway. Mutations in this part of fH might compromise its function and lead to underregulation of the alternative pathway. It is hypothesized that this can cause predisposition to diseases such as atypical haemolytic uraemic syndrome (aHUS) and age-related macular degeneration (AMD). In the current work, the known disease-associated mutations R53H and R78G were compared to wild-type in terms of fluid-phase cofactor assays, C3b-binding affinity and the ability to accelerate the decay of the convertase. In addition, the protective variant, I62, was also inspected because its protective role might be explained by an increased regulatory activity. The second, linked, aim of this project was to employ Forster resonance energy transfer (FRET) to study the link between conformation and function in fH. FRET is valuable for obtaining long-distance restraints up to a maximum of 100 °A and is therefore particularly useful for inferring domain orientations within multidomain proteins. This approach to measure long-range inter- and intramolecular distances is a convenient way to complement NMR-based structural investigations, which rely on short-range restraints. It is also a valuable complement to X-ray crystallography since it is a solution technique that can be conducted under physiological conditions. By using site-directed mutagenesis in the current work, free cysteines were introduced into CCP modules 1-4 at strategic points, which were then used for attachment of fluorescent tags. C3 possesses an internal thioester which can be labelled with a fluorophore upon activation to C3b. Intermolecular FRET measurements were thus undertaken to gain information about the interaction between the two proteins that is crucial for understanding functional activity. The CCP modules in the centre of fH may be responsible for introducing a bend into fH that brings the N-teminus close to the C-terminus (the latter is important for host versus non-host discrimination) joined by the longest linkers occurring in the whole molecule. This coincidence of two relatively small CCP modules, 12 and 13, with the highest number of eight amino acids between them, is hypothesised to reflect some unique architectural features. To explore the structural details of this portion of fH by FRET, single-labelled cysteine mutants were further modifed to provide a recognition site for transglutaminase (TGase), which can be enzymatically labeled with a second fluorophore. This stoichiometrically-labelled protein was used for intramolecular FRET studies.