Determination of three-bond 1H3'-31P couplings in nucleic acids and protein-nucleic acid complexes by quantitative J...

A new sensitive two-dimensional quantitative J correlation experiment is described for measuring 3JH3'-P couplings in nucleic acids and protein-nucleic acid complexes. The method is based on measuring the change in intensity of the 1H-1H cross peaks in a constant-time 1H-1H COSY experiment which occurs in the presence and absence of 3JH3'-P dephasing during the constant-time evolution period. For protein-nucleic acid complexes where the protein is 13C-labeled but the nucleic acid is not, 12C-filtering is readily achieved by the application of a series of 13C purge pulses during the constant time evolution period without any loss of signal-to-noise of the nucleic acid cross peaks. The method is demonstrated for the Dickerson DNA dodecamer and a 19 kDa complex of the transcription factor SRY with a 14mer DNA duplex. The same approach should be equally applicable to numerous other problems, including the measurement of JH-Cd couplings in cadmium-ligated proteins, or 3JCH couplings in other selectively enriched compounds.