Measurement of 1Hâ??15N and 1Hâ??13C residual dipolar couplings in nucleic acids from TROSY intensities
Measurement of 1Hâ??15N and 1Hâ??13C residual dipolar couplings in nucleic acids from TROSY intensities
Abstract Analogous to the recently introduced ARTSY method for measurement of one-bond 1Hâ??15N residual dipolar couplings (RDCs) in large perdeuterated proteins, we introduce methods for measurement of base 13Câ??1H and 15Nâ??1H RDCs in protonated nucleic acids. Measurements are based on quantitative analysis of intensities in 1Hâ??15N and 13Câ??1H TROSY-HSQC spectra, and are illustrated for a 71-nucleotide adenine riboswitch. Results compare favorably with those of conventional frequency-based measurements in terms of completeness and convenience of use. The ARTSY method derives the size of the coupling from the ratio of intensities observed in two TROSY-HSQC spectra recorded with different dephasing delays, thereby minimizing potential resonance overlap problems. Precision of the RDC measurements is limited by the signal-to-noise ratio, S/N, achievable in the 2D TROSY-HSQC reference spectrum, and is approximately given by 30/(S/N) Hz for 15Nâ??1H and 65/(S/N) Hz for 13Câ??1H. The signal-to-noise ratio of both 1Hâ??15N and 1Hâ??13C spectra greatly benefits when water magnetization during the experiments is not perturbed, such that rapid magnetization transfer from bulk water to the nucleic acid, mediated by rapid amino and hydroxyl hydrogen exchange coupled with 1Hâ??1H NOE transfer, allows for fast repetition of the experiment. RDCs in the mutated helix 1 of the riboswitch are compatible with nucleotide-specifically modeled, idealized A-form geometry and a static orientation relative to the helix 2/3 pair, which differs by ca 6° relative to the X-ray structure of the native riboswitch.
- Content Type Journal Article
- Category Article
- Pages 89-103
- DOI 10.1007/s10858-011-9544-y
- Authors
- Jinfa Ying, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 5, Room 126, Bethesda, MD 20892-0520, USA
- Jinbu Wang, Structural Biophysics Laboratory, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA
- Alex Grishaev, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 5, Room 126, Bethesda, MD 20892-0520, USA
- Ping Yu, Structural Biophysics Laboratory, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA
- Yun-Xing Wang, Structural Biophysics Laboratory, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA
- Ad Bax, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 5, Room 126, Bethesda, MD 20892-0520, USA
Source: Journal of Biomolecular NMR
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