Assignment of the 13C and 13CO resonances for Rhodobacter capsulatus ferrocytochrome c2 using double-resonance and triple-resonance NMR spectroscopy.

Abstract

Rhodobacter capsulatus cytochrome c2 uniformly labelled with 13C/15N has been prepared. The 13C resonances of the reduced state, including those of the carbonyl and heme 13C, have been assigned using a combination of various two- and three-dimensional correlated NMR experiments. Assignment of the sidechain 13C resonances facilitated correction of a small number of previously misassigned sidechain 1H and led to the additional assignment of 32 1H. It was found that 13C alpha and 13CO secondary shifts were better indicators of secondary structure than 1H alpha and 13C beta secondary shifts. Moreover, it was demonstrated that, despite the significant ring current effects present in heme proteins, 13C alpha and 13CO secondary shifts can be employed to accurately identify secondary structure in heme proteins, independently of NOE experiments.