Measurements of Ha-HN vicinal coupling constants in a protein with large line widths in a new 3D 1H-15N-13C quadruple resonance NMR experiment

P. Schmieder, V. Thanabal, L.P. McIntosh, F.W. Dahlquist, G. Wagner

J. Am. Chem. Soc. (1991) 113, 6323-6324

A 3D 1H-15N-13C quadruple resonance NMR experiment was developed that provides precise measurements of homonuclear Ha-HN coupling constants. in proteins. This approach requires that the proteins are enriched with both 15N and 13C. Experiments on the 164 residue protein T4 lysozyme are presented. The pulse sequence consists of a nonrefocused INEPT polarization transfer from 1H to 15N. This is followed by a 15N evolution period (t1). Afterwards, a 15N-13C heteronuclear multiple quantum evolution period yields labeling of the coherence with the Ca frequency during t2. The Ca-Hacoupling is active during t2. After refocussing, the 15N coherence is transferred to the HN via a TANGO pulse that is a 90° pulse selective for HN signals and does not mix the orientation of the Haspins. This leads to cross peaks in the w2-w3 planes that consist of two components that are separated by the one-bound Ha-Ca coupling along w2, and by the three-bond Ha-HN coupling along w3, which can readily be measured even in proteins with large line widths.