Direct 13C-detection for carbonyl relaxation studies of protein dynamics

We describe a method that uses direct C-13-detection for measuring rotating-frame carbonyl ((CO)-C-13) relaxation rates to describe protein functional dynamics. Key advantages of method include the following: (i) unique access to 13CO groups that lack a scalar-coupled N-15-H-1 group: (ii) insensitivity to N-15/H-1 exchange-broadening that can derail H-1-detected N-15 and HNCO methods: (iii) avoidance of artifacts caused by incomplete water Suppression. We demonstrate the approach for both backbone and side-chain 13CO groups. Accuracy of the C-13-detected results is Supported by their agreement with those obtained from established HNCO-based approaches. Critically, we show that the C-13-detection approach provides access to the 13CO groups of functionally important residues that are invisible via H-1-detected HNCO methods because of exchange-broadening. Hence, the C-13-based method fills gaps inherent in canonical H-1-detected relaxation experiments, and thus provides a novel complementary tool for NMR studies of biomolecular flexibility. (c) 2008 Elsevier Inc. All rights reserved.