MEASUREMENT OF H-N-H-ALPHA J-COUPLINGS IN CALCIUM-FREE CALMODULIN USING NEW 2D AND 3D WATER-FLIP-BACK METHODS

Two new methods are described for the measurement of three-bond J(HNH alpha) couplings in proteins isotopically enriched with N-15. Both methods leave the water magnetization in an unsaturated state, parallel to the z-axis, and therefore offer significant enhancements in sensitivity for rapidly exchanging backbone amide protons. The J couplings can be measured either from a set of constant-time 2D H-1-N-15 HMQC spectra, which are modulated in intensity by J(HNH alpha) or from a water-flip-back version of the 3D HNHA experiment. The method is demonstrated for a sample of calcium-free calmodulin. Residues Lys(75)-Asp(80) have J(HNH alpha) values in the 6-7 Hz range, suggesting that a break in the 'central helix' occurs at the same position as previously observed in solution NMR studies of Ca2+-ligated calmodulin.