Measurement of N-15-H-1 coupling constants in uniformly N-15-labeled proteins: Application to the photoactive yellow protein

A modified HNHB experiment is presented that allows the determination of J(NH) coupling constants directly from the ratio of cross-peak to diagonal-peak intensities. The experiment was applied to the photoactive yellow protein (PYP) and yielded the magnitude of 117 (3)J(NHbeta) coupling constants. In addition, 29 (3)J(NHalpha(i-1)) coupling constants could be measured, providing information about the backbone angle psi. These data, in conjunction with the magnitudes of the (3)J((HHalpha)-H-N) coupling constants obtained from the HNHA spectrum, effectively discriminate the two possibilities for the stereospecific assignment of the H-alpha resonances in glycine residues. For all eight glycine residues in PYP that were not subject to conformational averaging and had non-degenerate H-alpha resonance frequencies, the J-coupling data, together with limited NOE data, yielded the stereospecific assignment of the H-alpha resonances for these residues. In addition, reliable and precise phi,psi dihedral constraints were also derived for these residues from the J-coupling data.