MEASUREMENT OF 2-BOND AND 3-BOND C-13-H-1 J-COUPLINGS TO THE C-DELTA CARBONS OF LEUCINE RESIDUES IN STAPHYLOCOCCAL NUCLEASE

A new H-1-detected 3D NMR experiment is described that permits quantitative measurement of two- and three-bond C-13-H-1 couplings in proteins with selectively C-enriched methyl sites. The method is demonstrated for staphylococcal nuclease selectively [5,5 C-13]-labeled in all 11 leucine positions and ligated with thymidine 3',5'-biphosphate and Ca2+. Two- and three-bond C-13 methyl-proton couplings are reported and, together with the measured three-bond J(CalphaCdelta) in uniformly C-13-enriched staphylococcal nuclease, the chi2-angles and the stereospecific assignments of the C(delta) methyl group with respect to the prochiral beta-protons were determined. The same residues that were previously found to have high degrees of internal mobility on the basis of C-13 relaxation times have measured coupling constants that are indicative of motional averaging.