Determination of 3J(H3i′, Pi+1) and 3J(H5i′/5i",Pi) coupling constants in 13C-labeled nucleic acids using constant-time HMQC

A novel H-1-C-13 correlated two-dimensional experiment, CT-HMQC-J, for the measurement of three-bond proton-phosphorus coupling constants in C-13-labeled DNA is described, The experiment is based on the intensity difference of H-1-C-13 cross peaks in the presence and absence of the proton-phosphorus coupling interaction during the constant-time period in HMQC experiment. The (3)J(H, P) coupling constants can be easily extracted from the intensity ratios of the two experiments. The method has been applied to a uniformly C-13,N-15-labeled d(GGAGGAT) 7-mer DNA sample, The proton-phosphorus coupling constants determined from CT-HMQC-J, together with the other three-bond coupling constants, are used to determine beta and epsilon torsion angles. The introduction of beta and epsilon restraints has improved the convergence as well as the quality of d(GGAGGAT) structure. (C) 1999 Academic Press.