Measurement of one-bond C-13(alpha)-H-1(alpha) residual dipolar coupling constants in proteins by selective manipulation of (CH alpha)-H-alpha spins

We have developed new 2D and 3D experiments for the measurement of C-alpha-H-alpha residual dipolar coupling constants in C-13 and N-15 labelled proteins. Two experiments, 2D (HNCO)-(J-CA)NH and 3D (HN)CO-(J-CA)NH, sample the C-alpha-H-alpha splitting by means of C-alpha magnetization, while 2D (J-HACACO)NH and 3D J-HA(CACO)NH use H-alpha magnetization to achieve a similar result. In the 2D experiments the Coupling evolution is superimposed on the evolution of the N-15 chemical shifts and the IPAP principle is used to obtain H-1-N-15 HSQC-like spectra from which the splitting is determined. The use ora third dimension in 3D experiments reduces spectral overlap to the point where use of all IPAP scheme may not be necessary. The length of the sampling interval in the J-dimension of these experiments is dictated solely by the relaxation properties of C-alpha or H-alpha nuclei. This was made possible by the use of C-alpha selective pulses in combination with either a DPFGSE or modified BIRD pulses. Inclusion of these pulse sequence elements in the J-evolution periods removes unwanted spin-spin interactions. This allows prolonged sampling periods (similar to 25 ms) yielding higher precision C-alpha-H-alpha splitting determination than is achievable with existing frequency based methods. (c) 2006 Elsevier Inc. All rights reserved.