Direct 13C-detection for carbonyl relaxation studies of protein dynamics.
We describe a method that uses direct 13C-detection for measuring rotating-frame carbonyl (13CO) 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 15N-1H group; (ii) insensitivity to 15N/1H exchange-broadening that can derail 1H-detected 15N 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 13C-detected results is supported by their agreement with those obtained from established HNCO-based approaches. Critically, we show that the 13C-detection approach provides access to the 13CO groups of functionally important residues that are invisible via 1H-detected HNCO methods because of exchange-broadening. Hence, the 13C-based method fills gaps inherent in canonical 1H-detected relaxation experiments, and thus provides a novel complementary tool for NMR studies of biomolecular flexibility.
Department of Chemistry and Biochemistry, University of Notre Dame, 251 Nieuwland Science Hall, Notre Dame, IN 46556, USA.
Gabriela
G
Pasat
John S
JS
Zintsmaster
Jeffrey W
JW
Peng
D000465:Algorithms; D002247:Carbon Isotopes / Q000032:analysis / Q000737:chemistry*; D003198:Computer Simulation; D003461:Crystallography / Q000379:methods; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008956:Models, Chemical; D008958:Models, Molecular; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry* / Q000648:ultrastructure*
D002247:Carbon Isotopes; D011506:Proteins