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.
ASJC Scopus subject areas
- Nuclear and High Energy Physics
- Condensed Matter Physics