Measurement of one-bond 13Cα-1Hα residual dipolar coupling constants in proteins by selective manipulation of CαHα spins

We have developed new 2D and 3D experiments for the measurement of Cα-Hα residual dipolar coupling constants in 13C and 15N labelled proteins. Two experiments, 2D (HNCO)-(J-CA)NH and 3D (HN)CO-(J-CA)NH, sample the Cα-Hα splitting by means of Cα magnetization, while 2D (J-HACACO)NH and 3D J-HA(CACO)NH use Hα magnetization to achieve a similar result. In the 2D experiments the coupling evolution is superimposed on the evolution of the 15N chemical shifts and the IPAP principle is used to obtain 1H-15N HSQC-like spectra from which the splitting is determined. The use of a third dimension in 3D experiments reduces spectral overlap to the point where use of an 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 Cα or Hα nuclei. This was made possible by the use of Cα 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 (∼25 ms) yielding higher precision Cα-Hα splitting determination than is achievable with existing frequency based methods.