'1h nmr studies of plastocyanin in solution

This thesis describes the application of some modern techniques in nuclear magnetic resonance (NMR) spectroscopy to the `blue' copper redox protein plastocyanin. Two varieties of the protein, obtained from spinach and parsley leaves, with 99 and 97 amino acid residues respectively, have been investigated. Using sequence specific assignment methods the 1 H NMR spectrum of Cu(I) spinach plastocyanin has been essentially completely assigned. Using a variety of two-dimensional NMR techniques, including COSY, RELAY, DOUBLE RELAY and NOESY experiments, an assignment strategy was devised that enabled the sequential assignment to be successfully achieved, even though the amino acid spin systems with `long' side chains could not be fully resolved in the first stage assignment. The assignment of the spinach plastocyanin spectrum, made independently of any known structure, is then used to begin the modelling of the structure in solution. It is demonstrated that two extensive -sheets can be recognised from the pattern of long-range NOEs observed. Correlations are made, of the NMR data with the crystal structure of poplar plastocyanin, which has 22 amino acid differences in the sequence. No significant differences in the two structures have been discovered, either in the backbone fold or in the side chain interactions. The assignment of the spectrum of parsley plastocyanin has been initiated. Using a strategy identical to that used for spinach plastocyanin, approximately 95% of the backbone resonances have been identified. A model of a section of the backbone, which has two amino acid `deletions' in comparison with other plant plastocyanins, has been created. The model is based on the pattern of short- and long-range NOEs and slowly exchanging NH protons that have been observed in the spectrum. The effect of the `deletions' is to straighten a `kink' in the structure of poplar plastocyanin. The use of paramagnetic Cr(III) cations as a probe of the surface of plastocyanin has been explored both in 1D and 2D NMR experiments. It is demonstrated that, in the latter case, relaxation effects for the more hydrophilic surface side chains are more readily resolved. A large proportion of the surface of plastocyanin appears to be accessible to the cations, though it is clear that not all acidic side chains are involved with the association. The pattern of cation-binding effects is observed to be similar for both spinach and parsley plastocyanin, being centred on the aromatic ring of residue Tyr-83, and also for a variety of different cations. These results lead to the proposal of two hypothetical protein-cation binding scenarios. Finally, some consideration is given to extension of the use of paramagnetic probes in 2D NMR for the quantitative determination of binding interactions. (D76972)