Abstract:
Membrane proteins are poorly represented in the structural databases, despite the fact that approximately 30% of all open reading frames code for them. Investigations into the structure of three membrane proteins are reported in this thesis: OpcA, the outer membrane adhesin from the pathogenic bacterium Neisseria meningitidis; Kir2.1, the inwardly rectifying potassium channel from Mus musculus; PSD95, the post-synaptic density scaffold protein from Rattus norvegicus. Recombinant OpcA was isolated from a bacterial expression system, refolded from inclusion bodies and purified in the presence of detergent. The purified protein was used in several structure determination experiments: OpcA was found to adopt a micelle sharing dimeric state in solution by small angle X-ray scattering; the location of detergent within crystals of the protein was explored by low-resolution neutron crystallography; and ligand binding was investigated by high resolution X-ray crystallography experiments. Perdeuterated OpcA was also purified and crystallised, and was found to diffract neutrons during a test scan. Finally, the structures of Kir2.1 and PSD95 were investigated by small angle scattering. A cytoplasmic domain construct of Kir2.1 was found to be have a tetrameric structure. Rigid body modelling was performed on PSD95 using the experimental small angle scattering curve to fit the domains of the protein.
