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

A modified HNHB experiment is presented that allows thedetermination of J(NH) coupling constants directly from the ratio ofcross-peak to diagonal-peak intensities. The experiment was applied to thephotoactive yellow protein (PYP) and yielded the magnitude of 1173J(NHβ) coupling constants. In addition, 293J(NHα(i−1)) coupling constantscould be measured, providing information about the backbone angle ψ.These data, in conjunction with the magnitudes of the3J(HNHα) coupling constantsobtained from the HNHA spectrum, effectively discriminate the twopossibilities for the stereospecific assignment of theHα resonances in glycine residues. For all eight glycineresidues in PYP that were not subject to conformational averaging and hadnon-degenerate Hα resonance frequencies, the J-couplingdata, together with limited NOE data, yielded the stereospecific assignmentof the Hα resonances for these residues. In addition,reliable and precise φ,ψ dihedral constraints were also derived forthese residues from the J-coupling data.