QUANTITATIVE J CORRELATION - A NEW APPROACH FOR MEASURING HOMONUCLEAR3-BOND J(H(N)H(ALPHA) COUPLING-CONSTANTS IN N-15-ENRICHED PROTEINS

A new approach is described for the measurement of homonuclear H(N)-H( alpha) J couplings in N-15-enriched proteins. The method relies on mea surement of the diagonal-peak to cross-peak intensity ratio in a 3D N- 15-separated quantitative J-correlation spectrum. The experiment is de monstrated for the protein staphylococcal nuclease, uniformly enriched with N-15, ligated with thymidine 3'-5'-bisphosphate and Ca2+. Amide to C(alpha)H correlations are observed for all but three of the amide protons that have regular intensities in a 2D H-1-N-15 correlation spe ctrum, and J couplings could be measured for 96 residues with sufficie ntly resolved H-1-N-15 correlations, including 6 glycines. In addition , for 28 residues with partially overlapping H-1-N-15 correlations, co upling constants can be estimated as small, medium, or large. For 86 o ut of the 96 residues, the backbone angle phi is known with good preci sion from the crystal structure, and J values for these residues have a 0.73 Hz root-mean-square deviation from the Karplus curve: J = A cos 2(phi - 60) + B cos(phi - 60) + C, where A = 6.51, B = -1.76, C = 1.60 .