Accurate measurement of 15N–13C residual dipolar couplings in nucleic acids
Source: Journal of Biomolecular NMR, Volume 31, Number 3, March 2005 , pp. 231-241(11)
Abstract:New 3D HCN quantitative J (QJ) pulse schemes are presented for the precise and accurate measurement of one-bond 15N1/9–13C1′, 15N1/9–13C6/8, and 15N1/9–13C2/4 residual dipolar couplings (RDCs) in weakly aligned nucleic acids. The methods employ 1H–13C multiple quantum (MQ) coherence or TROSY-type pulse sequences for optimal resolution and sensitivity. RDCs are obtained from the intensity ratio of H1′–C1′–N1/9 (MQ-HCN-QJ) or H6/8–C6/8–N1/9 (TROSY-HCN-QJ) correlations in two interleaved 3D NMR spectra, with dephasing intervals of zero (reference spectrum) and ∼1/(2JNC) (attenuated spectrum). The different types of 15N–13C couplings can be obtained by using either the 3D MQ-HCN-QJ or TROSY-HCN-QJ pulse scheme, with the appropriate setting of the duration of the constant-time 15N evolution period and the offset of two frequency-selective 13C pulses. The methods are demonstrated for a uniformly 13C, 15N-enriched 24-nucleotide stem-loop RNA sequence, helix-35, aligned in the magnetic field using phage Pf1. For measurements of RDCs systematic errors are found to be negligible, and experiments performed on a 1.5 mM helix-35 sample result in an estimated precision of ca. 0.07 Hz for 1DNC, indicating the utility of the measured RDCs in structure validation and refinement. Indeed, for a complete set of 15N1/9–13C1′, 15N1/9–13C6/8, and 15N1/9–13C2/4 dipolar couplings obtained for the stem nucleotides, the measured RDCs are in excellent agreement with those predicted for an NMR structure of helix-35, refined using independently measured observables, including 13C–1H, 13C–13C and 1H–1H dipolar couplings.
Document Type: Research Article
Affiliations: 1: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 20892-0520, USA, 2: Laboratoire de RMN, Institut de Biologie Structurale, Jean-Pierre Ebel, UMR 5075, CNRS-CEA-UJF, 41 rue Jules Horowitz, 38027, Grenoble, Cedex, 1, France, 3: Department of Biochemistry and Cell Biology, Rice University, P.O. Box 1892, Houston, TX, 77251, USA,
Publication date: March 1, 2005