Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Measurement of residual dipolar couplings in methyl groups via carbon detection.

ABSTRACT: Residual dipolar couplings (RDCs) provide both structural and dynamical information useful in the characterization of biological macromolecules. While most data come from the interaction of simple pairs of directly bonded spin-1/2 nuclei (1H-15N, 1H-13C, 1H-1H), it is possible to acquire data from interactions among the multiple spins of 13C-labeled methyl groups (1H3-13C). This is especially important because of the advantages that observation of 13C-labeled methyl groups offers in working with very large molecules. Here we consider some of the options for measurement of methyl RDCs in large and often fully protonated proteins and arrive at a pulse sequence that exploits both J-modulation and direct detection of 13C. Its utility is illustrated by application to a fully protonated two domain fragment from the mammalian glycoprotein, Robo1, 13C-methyl-labeled in all valines.

SUBMITTER: Williams RV

PROVIDER: S-EPMC7020099 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Measurement of residual dipolar couplings in methyl groups via carbon detection.

Williams Robert V RV Yang Jeong-Yeh JY Moremen Kelley W KW Amster I Jonathan IJ Prestegard James H JH

Journal of biomolecular NMR 20190430 3-4

Residual dipolar couplings (RDCs) provide both structural and dynamical information useful in the characterization of biological macromolecules. While most data come from the interaction of simple pairs of directly bonded spin-1/2 nuclei (1H-15N, 1H-13C, 1H-1H), it is possible to acquire data from interactions among the multiple spins of 13C-labeled methyl groups (1H3-13C). This is espec ...[more]

PMID: 31041649

Similar Datasets

Measurement of imino 1H-1H residual dipolar couplings in RNA.

Project description:Imino (1)H-(15)N residual dipolar couplings (RDCs) provide additional structural information that complements standard (1)H-(1)H NOEs leading to improvements in both the local and global structure of RNAs. Here, we report measurement of imino (1)H-(1)H RDCs for the Iron Responsive Element (IRE) RNA and native E. coli tRNA(Val) using a BEST-Jcomp-HMQC2 experiment. (1)H-(1)H RDCs are observed between the imino protons in G-U wobble base pairs and between imino protons on neighboring base pairs in both RNAs. These imino (1)H-(1)H RDCs complement standard (1)H-(15)N RDCs because the (1)H-(1)H vectors generally point along the helical axis, roughly perpendicular to (1)H-(15)N RDCs. The use of longitudinal relaxation enhancement increased the signal-to-noise of the spectra by ~3.5-fold over the standard experiment. The ability to measure imino (1)H-(1)H RDCs offers a new restraint, which can be used in NMR domain orientation and structural studies of RNAs.

| S-EPMC2846714 | biostudies-literature

Measurement of Residual Dipolar Couplings Using Magnetically Aligned and Flipped Nanodiscs.

Project description:Recent developments in lipid nanodisc technology have successfully overcome the major challenges in the structural and functional studies of membrane proteins and drug delivery. Among the different types of nanodiscs, the use of synthetic amphiphilic polymers created new directions including the applications of solution and solid-state NMR spectroscopy. The ability to magnetically align large-size (>20 nm diameter) polymer nanodiscs and flip them using paramagnetic lanthanide ions has enabled high-resolution studies on membrane proteins using solid-state NMR techniques. The use of polymer-based macro-nanodiscs (>20 nm diameter) as an alignment medium to measure residual dipolar couplings (RDCs) and residual quadrupole couplings by NMR experiments has also been demonstrated. In this study, we demonstrate the use of magnetically aligned and 90°-flipped polymer nanodiscs as alignment media for structural studies on proteins by solution NMR spectroscopy. These macro-nanodiscs, composed of negatively charged SMA-EA polymers and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipids, were used to measure residual 1H-15N dipolar couplings (RDCs) from the water-soluble ∼21 kDa uniformly 15N-labeled flavin mononucleotide binding domain (FBD) of cytochrome-P450 reductase. The experimentally measured 1H-15N RDC values are compared with the values calculated from the crystal structures of cytochrome-P450 reductase that lacks the transmembrane domain. The N-H RDCs measured using aligned and 90°-flipped nanodiscs show a modulation by the function (3 cos2 θ - 1), where θ is the angle between the N-H bond vector and the applied magnetic field direction. This successful demonstration of the use of two orthogonally oriented alignment media should enable structural studies on a variety of systems including small molecules, DNA, and RNA.

| S-EPMC9575995 | biostudies-literature

Measurement and interpretation of 15N-1H residual dipolar couplings in larger proteins.

Project description:A decade ago, Dr. L.E. Kay and co-workers described an ingenious HNCO-based triple-resonance experiment from which several protein backbone RDCs can be measured simultaneously (Yang et al. (1999) [1]). They implemented a J-scaling technique in the (15)N dimension of the 3D experiment to obtain the NH RDCs. We have used this idea to carry out J-scaling in a 2D (15)N-(1)H-TROSY experiment and have found it to be an excellent method to obtain NH RDCs for larger proteins upto 70 kDa, far superior to commonly used HSQC in-phase/anti-phase and HSQC/TROSY comparisons. Here, this method, dubbed "RDC-TROSY" is discussed in detail and the limits of its utility are assessed by simulations. Prominent in the latter analysis is the evaluation of the effect of amide proton flips on the "RDC-TROSY" linewidths. The details of the technical and computational implementations of these methods for the determination of domain orientations in 45-60 kDa Hsp70 chaperone protein constructs are described.

| S-EPMC2835473 | biostudies-literature

Facile measurement of ¹H-¹5N residual dipolar couplings in larger perdeuterated proteins.

Project description:We present a simple method, ARTSY, for extracting ¹J(NH) couplings and ¹H-¹⁵N RDCs from an interleaved set of two-dimensional ¹H-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 characteristics, demonstrates its practical utility. Precision of the RDC measurement is limited by the signal-to-noise ratio, S/N, achievable in the 2D TROSY-HSQC spectrum, and is approximately given by 30/(S/N) Hz.

| S-EPMC2950907 | biostudies-literature

Measuring membrane protein bond orientations in nanodiscs via residual dipolar couplings.

Project description:Membrane proteins are involved in numerous vital biological processes. To understand membrane protein functionality, accurate structural information is required. Usually, structure determination and dynamics of membrane proteins are studied in micelles using either solution state NMR or X-ray crystallography. Even though invaluable information has been obtained by this approach, micelles are known to be far from ideal mimics of biological membranes often causing the loss or decrease of membrane protein activity. Recently, nanodiscs, which are composed of a lipid bilayer surrounded by apolipoproteins, have been introduced as a more physiological alternative than micelles for NMR investigations on membrane proteins. Here, we show that membrane protein bond orientations in nanodiscs can be obtained by measuring residual dipolar couplings (RDCs) with the outer membrane protein OmpX embedded in nanodiscs using Pf1 phage as an alignment medium. The presented collection of membrane protein RDCs in nanodiscs represents an important step toward more comprehensive structural and dynamical NMR-based investigations of membrane proteins in a natural bilayer environment.

| S-EPMC4088969 | biostudies-literature

Accurate Measurement of Residual Dipolar Couplings in Large RNAs by Variable Flip Angle NMR.

Project description:NMR approaches using nucleotide-specific deuterium labeling schemes have enabled structural studies of biologically relevant RNAs of increasing size and complexity. Although local structure is well-determined using these methods, definition of global structural features, including relative orientations of independent helices, remains a challenge. Residual dipolar couplings, a potential source of orientation information, have not been obtainable for large RNAs due to poor sensitivity resulting from rapid heteronuclear signal decay. Here we report a novel multiple quantum NMR method for RDC determination that employs flip angle variation rather than a coupling evolution period. The accuracy of the method and its utility for establishing interhelical orientations are demonstrated for a 36-nucleotide RNA, for which comparative data could be obtained. Applied to a 78 kDa Rev response element from the HIV-1 virus, which has an effective rotational correlation time of ca. 160 ns, the method yields sensitivity gains of an order of magnitude or greater over existing approaches. Solution-state access to structural organization in RNAs of at least 230 nucleotides is now possible.

| S-EPMC6021016 | biostudies-literature

Partial alignment and measurement of residual dipolar couplings of proteins under high hydrostatic pressure.

Project description:High-pressure NMR spectroscopy has emerged as a complementary approach for investigating various structural and thermodynamic properties of macromolecules. Noticeably absent from the array of experimental restraints that have been employed to characterize protein structures at high hydrostatic pressure is the residual dipolar coupling, which requires the partial alignment of the macromolecule of interest. Here we examine five alignment media that are commonly used at ambient pressure for this purpose. We find that the spontaneous alignment of Pf1 phage, d(GpG) and a C12E5/n-hexnanol mixture in a magnetic field is preserved under high hydrostatic pressure. However, DMPC/DHPC bicelles and collagen gel are found to be unsuitable. Evidence is presented to demonstrate that pressure-induced structural changes can be identified using the residual dipolar coupling.

| S-EPMC3758465 | biostudies-literature

Residual dipolar couplings: are multiple independent alignments always possible?

Project description: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 protein'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 alignment media, these data are suitable for structure refinement, but not the extraction of dynamic parameters. For an analysis of protein dynamics the data must be obtained with very low errors in at least three or five independent alignment media (depending on the method used) and so far, such data have only been reported for three small 6-8 kDa proteins with identical folds: ubiquitin, GB1 and GB3. Our results suggest that HEWL is likely to be representative of many other medium to large sized proteins commonly studied by solution NMR. Comparisons with over 60 high-resolution crystal structures of HEWL reveal that the highest resolution structures are not necessarily always the best models for the protein structure in solution.

| S-EPMC3020303 | biostudies-literature

A unifying probabilistic framework for analyzing residual dipolar couplings.

Project description:Residual dipolar couplings provide complementary information to the nuclear Overhauser effect measurements that are traditionally used in biomolecular structure determination by NMR. In a de novo structure determination, however, lack of knowledge about the degree and orientation of molecular alignment complicates the analysis of dipolar coupling data. We present a probabilistic framework for analyzing residual dipolar couplings and demonstrate that it is possible to estimate the atomic coordinates, the complete molecular alignment tensor, and the error of the couplings simultaneously. As a by-product, we also obtain estimates of the uncertainty in the coordinates and the alignment tensor. We show that our approach encompasses existing methods for determining the alignment tensor as special cases, including least squares estimation, histogram fitting, and elimination of an explicit alignment tensor in the restraint energy.

| S-EPMC2758374 | biostudies-literature

Determination of protein backbone structures from residual dipolar couplings.

Project description:There are a number of circumstances in which a focus on determination of the backbone structure of a protein, as opposed to a complete all-atom structure, may be appropriate. This is particularly the case for structures determined as a part of a structural genomics initiative in which computational modeling of many sequentially related structures from the backbone of a single family representative is anticipated. It is, however, also the case when the backbone may be a stepping-stone to more targeted studies of ligand interaction or protein-protein interaction. Here an NMR protocol is described that can produce a backbone structure of a protein without the need for extensive experiments directed at side chain resonance assignment or the collection of structural information on side chains. The procedure relies primarily on orientational constraints from residual dipolar couplings as opposed to distance constraints from NOEs. Procedures for sample preparation, data acquisition, and data analysis are described, along with examples from application to small target proteins of a structural genomics project.

| S-EPMC1808351 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data