| dc.description.abstract | 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. | en |
