Accurate measurement of N-15-C-13 residual dipolar couplings in nucleic acids

摘要

New 3D HCN quantitative J (QJ) pulse schemes are presented for the precise and accurate measurement of one-bond N-15(1/9)-C-13(1), N-15(1/9)-C-13(6/8), and N-15(1/9)-C-13(2/4) residual dipolar couplings (RDCs) in weakly aligned nucleic acids. The methods employ H-1-C-13 multiple quantum (MQ) coherence or TROSY-type pulse sequences for optimal resolution and sensitivity. RDCs are obtained from the intensity ratio of H-1,-C-1'-N-1/9 (MQ-HCN-QJ) or H-6/8-C-6/8-N-1/9 (TROSY-HCN-QJ) correlations in two interleaved 3D NMR spectra, with dephasing intervals of zero (reference spectrum) and ∼ 1/(2J(NC)) (attenuated spectrum). The different types of N-15-C-13 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 N-15 evolution period and the offset of two frequency-selective C-13 pulses. The methods are demonstrated for a uniformly C-13, N-15-enriched 24-nucleotide stem-loop RNA sequence, helix-35ψ, 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-35ψ sample result in an estimated precision of ca. 0.07 Hz for D-1(NC), indicating the utility of the measured RDCs in structure validation and refinement. Indeed, for a complete set of N-15(1/9)-C-13(1'), N-15(1/9)-C-13(6/8), and N-15(1/9)-C-13(2/4) dipolar couplings obtained for the stem nucleotides, the measured RDCs are in excellent agreement with those predicted for an NMR structure of helix-35ψ, refined using independently measured observables, including C-13-H-1, C-13-C-13 and H-1-H-1 dipolar couplings.