The effects of multiple amino acid substitutions on the polypeptide backbone of tuna and horse cytochromes c.

The cytochromes c provide a wide range of natural and mutant homologous proteins which may be used to study structure/function relationships in biological electron-transfer reactions. A description of the cytochrome c structure has been provided by high-resolution X-ray crystallography for the cytochromes from tuna, bonito, rice and yeast (Saccharomyces iso-1). Correlation of these structures with NMR parameters is necessary to confirm the structure of the protein in solution and to permit the routine characterisation of cytochromes c with novel sequences. We have previously reported a method based on the analysis of pseudocontact shifts which allowed us to compare the conformations of some amino acid side chains of tuna cytochrome c in solution and in the crystalline state. Here we report a comparison of the conformations of the polypeptide backbone of cytochromes c in proteins from tuna and horse, using the chemical shifts of the amide NH and C alpha H protons. It is found that the backbone conformation and hydrogen-bond network is closely conserved between these proteins, despite 19 amino acid substitutions. Appreciable differences occur in two regions, around Asn 31 and at the beginning of the 60s helix. Evidence for some rotational or translational motion of the C-terminal helix is also presented.