Proton resonance assignments of horse ferrocytochrome c

Abstract

Two-dimensional nuclear magnetic resonance (NMR) spectroscopy was used to assign the proton resonances of horse ferrocytochrome c. Assignments were based on the main chain directed (MCD) and sequential assignment procedures. The fundamental units of the MCD approach, the main-chain NH-C alpha H-C beta H J-coupled subspin systems of each amino acid residue (NAB sets), were defined by analysis of direct and relayed coherence transfer spectra. Recognition of main-chain NOE connectivity patterns specified in the MCD algorithm then allowed NAB sets to be aligned in their proper juxtaposition within secondary structural units. The units of secondary structure were placed within the polypeptide sequence of identification of a small number of side-chain J-coupled spin systems, found by direct recognition in 2D spectra of some J-coupled spin systems and by pairwise comparisons of the J-correlated spectra of six homologous cytochromes c having a small number of known amino acid differences. The placement of a given segment in this way defines the amino acid identity of all its NAB sets. This foreknowledge allowed the vast majority of the side-chain resonances to be discerned in J-correlated spectra. Extensive confirmation of the assignments derives internally from multiple main-chain NOE connectivities and their consistency following temperature-induced changes of the chemical shifts of NOE-correlated protons. The observed patterns of main-chain NOEs provide some structural information and suggest small but potentially significant differences between the solution structure observed by NMR and that defined earlier in crystallographic studies at 2.8-A resolution.