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
Simultaneous measurement of 1Hâ??15N and Methyl 1Hmâ??13Cm residual dipolar couplings in large proteins
Simultaneous measurement of 1Hâ??15N and Methyl 1Hmâ??13Cm residual dipolar couplings in large proteins
Abstract A two-dimensional TROSY-based SIM-13Cmâ??1Hm/1Hâ??15N NMR experiment for simultaneous measurements of methyl 1 D CH and backbone amide 1 D NH residual dipolar couplings (RDC) in {U-; Ileδ1-; Leu,Val-}-labeled samples of large proteins is described. Significant variation in the alignment tensor of the 82-kDa enzyme Malate synthase G is observed as a function of only slight changes in experimental conditions. The SIM-13Cmâ??1Hm/1Hâ??15N data sets provide convenient means...
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09-30-2011 08:01 PM
Rapid measurement of residual dipolar couplings for fast fold elucidation of proteins
Rapid measurement of residual dipolar couplings for fast fold elucidation of proteins
Abstract It has been demonstrated that protein folds can be determined using appropriate computational protocols with NMR chemical shifts as the sole source of experimental restraints. While such approaches are very promising they still suffer from low convergence resulting in long computation times to achieve accurate results. Here we present a suite of time- and sensitivity optimized NMR experiments for rapid measurement of up to six RDCs per residue. Including such an RDC data set, measured in less...
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09-17-2011 10:20 AM
Residual dipolar couplings: are multiple independent alignments always possible?
Residual dipolar couplings: are multiple independent alignments always possible?
Abstract RDCs for the 14 kDa protein hen egg-white lysozyme (HEWL) have been measured in eight different alignment media. The elongated shape and strongly positively charged surface of HEWL appear to limit the protein to four main alignment orientations. Furthermore, low levels of alignment and the proteinâ??s interaction with some alignment media increases the experimental error. Together with heterogeneity across the alignment media arising from constraints on temperature, pH and ionic strength for some...
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12-26-2010 04:43 AM
[NMR paper] Residual dipolar couplings in NMR structure analysis.
Residual dipolar couplings in NMR structure analysis.
Related Articles Residual dipolar couplings in NMR structure analysis.
Annu Rev Biophys Biomol Struct. 2004;33:387-413
Authors: Lipsitz RS, Tjandra N
Residual dipolar couplings (RDCs) have recently emerged as a new tool in nuclear magnetic resonance (NMR) with which to study macromolecular structure and function in a solution environment. RDCs are complementary to the more conventional use of NOEs to provide structural information. While NOEs are local-distance restraints, RDCs provide...
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11-24-2010 09:25 PM
[NMR paper] Residual dipolar couplings: synergy between NMR and structural genomics.
Residual dipolar couplings: synergy between NMR and structural genomics.
Related Articles Residual dipolar couplings: synergy between NMR and structural genomics.
J Biomol NMR. 2002 Jan;22(1):1-8
Authors: Al-Hashimi HM, Patel DJ
Structural genomics is on a quest for the structure and function of a significant fraction of gene products. Current efforts are focusing on structure determination of single-domain proteins, which can readily be targeted by X-ray crystallography, NMR spectroscopy and computational homology modeling. However,...
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11-24-2010 08:49 PM
Facile measurement of 1Hâ??15N residual dipolar couplings in larger perdeuterated pro
Abstract We present a simple method, ARTSY, for extracting 1JNH couplings and 1Hâ??15N RDCs from an interleaved set of two-dimensional 1Hâ??15N TROSY-HSQC spectra, based on the principle of quantitative J correlation. The primary advantage of the ARTSY method over other methods is the ability to measure couplings without scaling peak positions or altering the narrow line widths characteristic of TROSY spectra. Accuracy of the method is demonstrated for the model system GB3. Application to the catalytic core domain of HIV integrase, a 36 kDa homodimer with unfavorable spectral...
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08-14-2010 04:19 AM
MQ-HNCO-TROSY for the measurement of scalar and residual dipolar couplings in larger
Abstract We describe a novel pulse sequence, MQ-HNCO-TROSY, for the measurement of scalar and residual dipolar couplings between amide proton and nitrogen in larger proteins. The experiment utilizes the whole 2TN polarization transfer delay for labeling of 15N chemical shift in a constant time manner, which efficiently doubles the attainable resolution in 15N dimension with respect to the conventional HNCO-TROSY experiment. In addition, the accordion principle is employed for measuring (J + D)NHs, and the multiplet components are selected with the generalized version of the TROSY scheme...
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Composite Alignment Media for the Measurement of Independent Sets of NMR Residual Dipolar Couplings
Composite Alignment Media for the Measurement of Independent Sets of NMR Residual Dipolar Couplings
Ke Ruan and Joel R. Tolman
J. Am. Chem. Soc.; 2005; 127(43) pp 15032 - 15033;
Abstract:
The measurement of independent sets of NMR residual dipolar couplings (RDCs) in multiple alignment media can provide a detailed view of biomolecular structure and dynamics, yet remains experimentally challenging. It is demonstrated here that independent sets of RDCs can be measured for ubiquitin using just a single alignment medium composed of aligned bacteriophage Pf1 particles embedded in a...
Measurement of 1Hâ??15N and 1Hâ??13C residual dipolar couplings in nucleic acids from TROSY intensities
Linear Mode
