Efficient and precise measurement of H(alpha)-C(alpha), C(alpha)-C', C(alpha)-C(beta) and H(N)-N residual dipolar couplings from 2D H(N)-N correlation spectra.

A suite of experiments are presented for the measurement of H(alpha)-C(alpha), C(alpha)-C', C(alpha)-C(beta) and H(N)-N couplings from uniformly 15N, 13C labeled proteins. Couplings are obtained from a series of intensity modulated two-dimensional H(N)-N spectra equivalent to the common 1H-15N-HSQC spectra, alleviating many overlap and assignment issues associated with other techniques. To illustrate the efficiency of this method, H(alpha)-C(alpha), C(alpha)-C', and H(N)-N isotropic scalar couplings were determined for ubiquitin from data collected in less than 4.5 h, C(alpha)-C(beta) data collection required 10 h. The resulting couplings were measured with an average error of +/-0.06, +/-0.05, +/-0.04 and +/-0.10 Hz, respectively. This study also shows H(alpha)-C(alpha) and C(alpha)-C(beta) couplings, valuable because they provide orientation of bond vectors outside the peptide plane, can be measured in a uniform and precise way. Superior accuracy and precision to existing 3D measurements for C(alpha)-C' couplings and increased precision compared to IPAP measurements for H(N)-N couplings are demonstrated. Minor modifications allow for acquisition of modulated H(N)-C' 2D spectra, which can yield additional well resolved peaks and significantly increase the number of measured RDCs for proteins with crowded 1H-15N resonances.