A 3D triple resonance experiment has been designed to provide intraresidual and sequential correlations between amide nitrogens and c~-carbons in uniformly 13C/15N-labeled proteins. In-phase 13C~ 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 anaino acid type as well as establishing sequential connectivities. Leaving the carbonyl spins untouched throughout the transfer from t3C~ to 1H~ leads to E.COSY-type cross peaks, from which the 3Jitl3co coupling constants can be evaluated. The pulse sequence is applied to oxidized Desulfovibrio vulgaris flavodoxin.
The resonance assignment of a protein provides the basis for the interpretation of structural data, derived from multidimensional NMR experiments. As a first step, the approach generally applied for 13C/15N-labeled proteins leads to the assignment of all backbone resonances. The identification of spin systems is mainly performed in subsequent experiments, without direct correlation between backbone and side-chain resonances. In this communication we present a triple resonance 3D NMR technique which correlates the backbone ISN and 13C~ resonances with the aliphatic side-chain IH resonances in ~SN/13C-labeled proteins. In contrast to the previously published HCC(CO)NH-type experiments (