Complete 13C assignments for recombinant Cu(I) rusticyanin. Prediction of secondary structure from patterns of chemical shifts

Complete resonance assignments for the 13C spectrum of reduced (Cu(I)) rusticyanin have been made using 13C, 15N doubly labeled recombinant material. The reported assignments include those for the carboxyl and carbonyl carbon atoms and protonated aromatic ring carbons, and were obtained using a variety of 2- and 3D inverse-detected NMR experiments, including 13C, 15N, 1H triple resonance experiments and HCCH-COSY and -TOCSY. Backbone carbonyl assignments were obtained using 3D HNCO and HCACO spectra, and modified versions of 2D H(CA)CO and HMBC spectra were used to obtain side-chain carboxyl carbon and methionine epsilon-methyl carbon assignments, respectively. A comparison of the 13C alpha, 13C beta and 13CO chemical shifts with published 'random coil' values confirms the conclusion reached from a consideration of the 3JHN alpha coupling constants and the pattern of sequential NOEs, that the protein consists largely of beta-structure.