Determination of Three-Bond ~1H3'-~(31)P Couplings in Nucleic Acids and Protein-Nucleic Acid Complexes by Quantitative J Correlation Spectroscopy

摘要

A new sensitive two-dimensional quantitative J correlation experiment is described for measuring ~3J_(H3'-P) coupling in nuclei acids and protein-nuclei 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 ~3J_(H3'-P) dephasing during the constant-time evolution period. For protein-nucleic acid complexes where the protein is ~(13)C-labeled but the nucleic acid is not, ~(12)C-filtering is readily achieved by the application of a series of ~(13)C purge pulses during the constant time evolution period without any loss of signal-to-noise of the nuclei 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 J_(H-Cd) couplings in cadmium-ligand proteins, or ~3J_(CH) couplings in other selectively enriched compounds.