Determination of three-bond 1H3'-31P couplings in nucleic acids and protein-nucleic acid complexes by quantitative J correlation spectroscopy.
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.
Clore
G M
GM
National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 5, Bethesda, Maryland, 20892-0520, USA.
Murphy
E C
EC
Gronenborn
A M
AM
Bax
A
A
D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D009584:Nitrogen; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D009687:Nuclear Proteins / Q000737:chemistry; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D010758:Phosphorus; D011487:Protein Conformation; D051876:Sex-Determining Region Y Protein; D014157:Transcription Factors
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D051876:Sex-Determining Region Y Protein; D014157:Transcription Factors; D010758:Phosphorus; D004247:DNA; D009584:Nitrogen