An efficient NMR experiment for analyzing sugar-puckering in unlabeled DNA: application to the 26-kDa dead ringer-DNA complex.
We present a new NMR experiment for estimating the type and degree of sugar-puckering in high-molecular-weight unlabeled DNA molecules. The experiment consists of a NOESY sequence preceded by a constant-time scalar coupling period. Two subexperiments are compared, each differing in the amount of time the (3)J(H3'H2') and (3)J(H3'H2") couplings are active on the H3' magnetization. The resultant data are easy to analyze, since a comparison of the signal intensities of any resolved NOE cross peak originating from H3' atoms of the duplex can be used to estimate the sum of the (3)J(H3'H2') and (3)J(H3'H2") couplings and thus the puckering type of the deoxyribose ring. Isotope filters to eliminate signals of the (13)C-labeled component in the F1-dimension are implemented, facilitating analyses of high-molecular-weight protein-DNA complexes containing (13)C-labeled protein and unlabeled DNA. The utility of the experiment is demonstrated on the 26-kDa Dead Ringer protein-DNA complex and reveals that the DNA uniformly adopts the S-type configuration when bound to protein.
Iwahara
J
J
Department of Chemistry and Biochemistry, UCLA-DOE Laboratory of Structural Biology and Molecular Medicine, University of California, Los Angeles, 405 Hilgard Avenue, California 90095-1570, USA. iwahara@mbi.ucla.edu
Wojciak
J M
JM
Clubb
R T
RT
D002247:Carbon Isotopes; D004247:DNA / Q000737:chemistry*; D003855:Deoxyribose / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011506:Proteins / Q000737:chemistry
D002247:Carbon Isotopes; D011506:Proteins; D003855:Deoxyribose; D004247:DNA