1H-NMR sequential assignments and cation-binding studies of spinach plastocyanin

The essentially complete assignment of the 1H-NMR spectrum of the Cu(i) form of spinach plastocyanin has been achieved using two-dimensional NMR techniques and sequence-specific resonance assignment procedures. A variety of pH and temperature conditions was utilised to overcome the problems of resonance overlap in the spectrum, degeneracy of C alpha H and solvent H2O chemical shifts, and cross-saturation of labile NH resonances. A qualitative analysis of the long-range nuclear Overhauser effects observed indicates that the backbone fold of spinach plastocyanin is very similar to that of poplar plastocyanin, whose structure has been solved by X-ray crystallography and differs in 22 of its 99 amino acid residues. The assignments provide a basis for further investigations into the structural and ion- and protein-binding properties of plastocyanin in solution.