Nuclear magnetic resonance studies of modified eukaryotic cytochrome C
The central theme of this thesis is a study of the structural changes accompanying chemical modification and denaturation of eukaryotic cytochrome c as characterised by 1II nuclear magnetic resonance (n.m.r.) spectroscopy. First, however, it was necessary to obtain and confirm assignments for individual resonances; this was achieved by a novel method of cross assignment between ferricytochrome c and ferrocytochrome c and by double resonance techniques. A variety of perturbations were caused to native cytochromes c, which ranged in degree from the elevation of temperature for ferrocytochrome c to the complete denaturation of the protein with urea or methanol. Modification at single sites both on the surface (e.g. Met 65, Tyr 74) and in the core ( e.g. Tyr 67) of the molecule were found to cause only small local effects to the structure, although the dynamic features of the molecules were altered. One single site modification, the breaking of the iron - sulphur cross linking bond, caused considerable disruption to one side of the molecule, although hydrophobic domains in the other side were preserved; this state of the molecule is analogous to the penultimate state in the refolding pathway. Modification of all the charged lysine residues caused small changes to the surface structure of the molecule, though the complete reversal of the charges in maleyl cytochrome c produced a species which unfolded reversibly from a native configuration with the increase of temperature. The unfolding of the protein is virtually identical with both methanol and urea, but the pathways are shown to differ for the oxidised and reduced proteins.