Measurement of 1H–15N and 1H–13C residual dipolar couplings in nucleic acids from TROSY intensities
- 316 Downloads
Analogous to the recently introduced ARTSY method for measurement of one-bond 1H–15N residual dipolar couplings (RDCs) in large perdeuterated proteins, we introduce methods for measurement of base 13C–1H and 15N–1H RDCs in protonated nucleic acids. Measurements are based on quantitative analysis of intensities in 1H–15N and 13C–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 15N–1H and 65/(S/N) Hz for 13C–1H. The signal-to-noise ratio of both 1H–15N and 1H–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 1H–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.
KeywordsARTSY BEST A-form RNA Quantitative J correlation TROSY RDC RNA DNA
This work was supported by the Intramural Research Programs of the NIDDK and NCI, NIH, and by the Intramural AIDS-Targeted Antiviral Program of the Office of the Director, NIH.
- Pervushin K, Riek R, Wider G, Wuthrich K (1997) Attenuated T-2 relaxation by mutual cancellation of dipole- dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc Natl Acad Sci USA 94:12366–12371ADSCrossRefGoogle Scholar
- Skrynnikov NR, Goto NK, Yang DW, Choy WY, Tolman JR, Mueller GA, Kay LE (2000) Orienting domains in proteins using dipolar couplings measured by liquid-state NMR: Differences in solution and crystal forms of maltodextrin binding protein loaded with beta-cyclodextrin. J Mol Biol 295:1265–1273CrossRefGoogle Scholar
- Tolbert BS, Miyazaki Y, Barton S, Kinde B, Starck P, Singh R, Bax A, Case DA, Summers MF (2010) Major groove width variations in RNA structures determined by NMR and impact of C-13 residual chemical shift anisotropy and H-1-C-13 residual dipolar coupling on refinement. J Biomol NMR 47:205–219CrossRefGoogle Scholar
- Yang DW, Venters RA, Mueller GA, Choy WY, Kay LE (1999) TROSY-based HNCO pulse sequences for the measurement of (HN)-H- 1-N-15, N-15-(CO)-C-13, (HN)-H-1-(CO)-C-13, (CO)-C-13-C- 13(alpha) and (HN)-H-1-C-13(alpha) dipolar couplings in N-15, C-13, H-2-labeled proteins. J Biomol NMR 14:333–343CrossRefGoogle Scholar