H. Kuboniwa et al., MEASUREMENT OF H-N-H-ALPHA J-COUPLINGS IN CALCIUM-FREE CALMODULIN USING NEW 2D AND 3D WATER-FLIP-BACK METHODS, Journal of biomolecular NMR, 4(6), 1994, pp. 871-878
Two new methods are described for the measurement of three-bond J(HNH
alpha) couplings in proteins isotopically enriched with N-15. Both met
hods leave the water magnetization in an unsaturated state, parallel t
o the z-axis, and therefore offer significant enhancements in sensitiv
ity 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 spect
ra, which are modulated in intensity by J(HNH alpha) or from a water-f
lip-back version of the 3D HNHA experiment. The method is demonstrated
for a sample of calcium-free calmodulin. Residues Lys(75)-Asp(80) hav
e 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.