HNCCH-TOCSY, A TRIPLE-RESONANCE EXPERIMENT FOR THE CORRELATION OF BACKBONE C-13(ALPHA) AND N-15 RESONANCES WITH ALIPHATIC SIDE-CHAIN PROTON RESONANCES AND FOR MEASURING VICINAL (CO)-C-13,H-1(BETA) COUPLING-CONSTANTS IN PROTEINS

A 3D triple resonance experiment has been designed to provide intraresidual and sequential correlations between amide nitrogens and alpha-carbons in uniformly C-13/N-15-labeled proteins. In-phase C-13(alpha) magnetization is transferred to the aliphatic side-chain protons via the side-chain carbons using a CC-TOCSY mixing sequence. Thus, the experiment alleviates the resonance assignment process by providing information about the amino acid type as well as establishing sequential connectivities. Leaving the carbonyl spins untouched throughout the transfer from C-13 alpha to H-1(beta) leads to E.COSY-type cross peaks, from which the (3)J(H beta CO) coupling constants can be evaluated. The pulse sequence is applied to oxidized Desulfovibrio vulgaris flavodoxin.