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Determination of Three-Bond 1H3′–31P Couplings in Nucleic Acids and Protein–Nucleic Acid Complexes by Quantitative J Correlation Spectroscopy

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Abstract:

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.

Document Type: Miscellaneous

Affiliations: National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 5, Bethesda, Maryland, 20892-0520

Publication date: September 1, 1998

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