Two-dimensional proton NMR studies of cytochrome c: assignment of the N-terminal helix

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Abstract
The 1H resonances of 11 sequential amino acids in the N-terminal helix of horse ferrocytochrome c were studied by two-dimensional nuclear magnetic resonance techniques. All the main-chain protons from Lys-5 through Ala-15 and many of the side-chain proteins were assigned. J-Correlated spectroscopy (COSY) was used to distinguish protons on neighboring bonds and to recognize amino acid types. Nuclear Overhauser effect spectroscopy (NOESY) was used to define spatially contiguous protons and to determine amino acid sequence neighbors. The relayed coherence experiment (relay COSY) was used to resolve many ambiguities in intraresidue J-coupled connectivities and interresidue NOE connectivities. This required no explicit knowledge of the solution structure. The pattern of NOEs found is consistent with a regular .alpha. helix between glycine-6 and lysine-13; H bonding continues at least through alanine-15 [see Wand, A. J., Roder, H., and Englander. S. W. (1986) Biochemistry (following paper in this issue)]. Chain dosorder occurs at the N-terminus. There is no indication of significant spin diffusion among the backbone amide and .alpha.-protons of this 12.4-kilodalton protein even at the longest NOE mixing time used (140 ms).