Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Complexity of protein energy landscapes studied by solution NMR relaxation dispersion experiments.

ABSTRACT: The millisecond time scale motions in ribonuclease A (RNase A) were studied by solution NMR CPMG and off-resonance R1? relaxation dispersion experiments over a wide pH and temperature range. These experiments identify three separate protein regions termed Cluster 1, Cluster 2, and R33, whose motions are governed by distinct thermodynamic parameters. Moreover, each of these regions has motions with different pH dependencies. Cluster 1 shows an increase in activation enthalpy and activation entropy as the pH is lowered, whereas Cluster 2 exhibits the opposite behavior. In contrast, the activation enthalpy and entropy of R33 show no pH dependence. Compounding the differences, ?? values for Cluster 2 are characteristic of two-site conformational exchange, yet similar analysis for Cluster 1 indicates that this region of the enzyme exhibits conformational fluctuations between a major conformer and a pH-dependent average of protonated and deprotonated minor conformers.

SUBMITTER: Khirich G

PROVIDER: S-EPMC4710144 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Complexity of protein energy landscapes studied by solution NMR relaxation dispersion experiments.

Khirich Gennady G Loria J Patrick JP

The journal of physical chemistry. B 20150220 9

The millisecond time scale motions in ribonuclease A (RNase A) were studied by solution NMR CPMG and off-resonance R1ρ relaxation dispersion experiments over a wide pH and temperature range. These experiments identify three separate protein regions termed Cluster 1, Cluster 2, and R33, whose motions are governed by distinct thermodynamic parameters. Moreover, each of these regions has motions with different pH dependencies. Cluster 1 shows an increase in activation enthalpy and activation entrop ...[more]

PMID: 25680027

Similar Datasets

Probing excited states and activation energy for the integral membrane protein phospholamban by NMR CPMG relaxation dispersion experiments.

Project description:Phospholamban (PLN) is a dynamic single-pass membrane protein that inhibits the flow of Ca(2+) ions into the sarcoplasmic reticulum (SR) of heart muscle by directly binding to and inhibiting the SR Ca(2+)ATPase (SERCA). The PLN monomer is the functionally active form that exists in equilibrium between ordered (T state) and disordered (R state) states. While the T state has been fully characterized using a hybrid solution/solid-state NMR approach, the R state structure has not been fully portrayed. It has, however, been detected by both NMR and EPR experiments in detergent micelles and lipid bilayers. In this work, we quantitatively probed the mus to ms dynamics of the PLN excited states by observing the T state in DPC micelles using CPMG relaxation dispersion NMR spectroscopy under functional conditions for SERCA. The (15)N backbone and (13)C(delta1) Ile-methyl dispersion curves were fit using a two-state equilibrium model, and indicate that residues within domain Ia (residues 1-16), the loop (17-22), and domain Ib (23-30) of PLN undergo mus-ms dynamics (k(ex)=6100+/-800 s(-1) at 17 degrees C). We measured k(ex) at additional temperatures, which allowed for a calculation of activation energy equal to approximately 5 kcal/mol. This energy barrier probably does not correspond to the detachment of the amphipathic domain Ia, but rather the energy needed to unwind domain Ib on the membrane surface, likely an important mechanism by which PLN converts between high and low affinity states for its binding partners.

| S-EPMC3328355 | biostudies-literature

Accelerating 2D NMR relaxation dispersion experiments using iterated maps.

Project description:NMR relaxation dispersion experiments play a central role in exploring molecular motion over an important range of timescales, and are an example of a broader class of multidimensional NMR experiments that probe important biomolecules. However, resolving the spectral features of these experiments using the Fourier transform requires sampling the full Nyquist grid of data, making these experiments very costly in time. Practitioners often reduce the experiment time by omitting 1D experiments in the indirectly observed dimensions, and reconstructing the spectra using one of a variety of post-processing algorithms. In prior work, we described a fast, Fourier-based reconstruction method using iterated maps according to the Difference Map algorithm of Veit Elser (DiffMap). Here we describe coDiffMap, a new reconstruction method that is based on DiffMap, but which exploits the strong correlations between 2D data slices in a pseudo-3D experiment. We apply coDiffMap to reconstruct dispersion curves from an [Formula: see text] relaxation dispersion experiment, and demonstrate that the method provides fast reconstructions and accurate relaxation curves down to very low numbers of sparsely-sampled data points.

| S-EPMC7370911 | biostudies-literature

Geometric Approximation: A New Computational Approach To Characterize Protein Dynamics from NMR Adiabatic Relaxation Dispersion Experiments.

Project description:A new computational strategy is reported that provides a fast approximation of numerical solutions of differential equations in general. The method is demonstrated with the analysis of NMR adiabatic relaxation dispersion experiments to reveal biomolecular dynamics. When an analytical solution to the theoretical equations describing a physical process is not available, the new approach can significantly accelerate the computational speed of the conventional numerical integration up to 10(5) times. NMR adiabatic relaxation dispersion experiments enhanced with optimized proton-decoupled pulse sequences, although extremely powerful, have previously been refractory to quantitative analysis. Both simulations and experimental validation demonstrate detectable "slow" (microsecond to millisecond) conformational exchange rates from 10(2) to 10(5) s(-1). This greatly expanded time-scale range enables the characterization of a wide array of conformational fluctuations for individual residues, which correlate with biomolecular function and were previously inaccessible. Moreover, the new computational method can be potentially generalized for analysis of new types of relaxation dispersion experiments to characterize the various dynamics of biomolecular systems.

| S-EPMC6344943 | biostudies-literature

Mapping the energy landscape of protein-ligand binding <i>via</i> linear free energy relationships determined by protein NMR relaxation dispersion.

Project description:Biochemical signaling is mediated by complexes between macromolecular receptors and their ligands, with the duration of the signal being directly related to the lifetime of the ligand-receptor complex. In the field of drug design, the recognition that drug efficacy in vivo depends on the lifetime of the drug-protein complex has spawned the concept of designing drugs with particular binding kinetics. To advance this field it is critical to investigate how the molecular details of designed ligands might affect the binding kinetics, as well as the equilibrium binding constant. Here we use protein NMR relaxation dispersion to determine linear free energy relationships involving the on- and off-rates and the affinity for a series of congeneric ligands targeting the carbohydrate recognition domain of galectin-3. Using this approach we determine the energy landscape and the position of the transition state along the reaction coordinate of protein-ligand binding. The results show that ligands exhibiting reduced off-rates achieve this by primarily stabilizing the bound state, but do not affect the transition state to any greater extent. The transition state forms early, that is, it is located significantly closer to the free state than to the bound state, suggesting a critical role of desolvation. Furthermore, the data suggest that different subclasses of ligands show different behavior with respect to these characteristics.

| S-EPMC8341105 | biostudies-literature

Relaxation dispersion NMR spectroscopy for the study of protein allostery.

Project description:Allosteric transmission of information between distant sites in biological macromolecules often involves collective transitions between active and inactive conformations. Nuclear magnetic resonance (NMR) spectroscopy can yield detailed information on these dynamics. In particular, relaxation dispersion techniques provide structural, dynamic, and mechanistic information on conformational transitions occurring on the millisecond to microsecond timescales. In this review, we provide an overview of the theory and analysis of Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR experiments and briefly describe their application to the study of allosteric dynamics in the homeodomain from the PBX transcription factor (PBX-HD). CPMG NMR data show that local folding (helix/coil) transitions in one part of PBX-HD help to communicate information between two distant binding sites. Furthermore, the combination of CPMG and other spin relaxation data show that this region can also undergo local misfolding, reminiscent of conformational ensemble models of allostery.

| S-EPMC5425744 | biostudies-literature

Longitudinal-relaxation-enhanced NMR experiments for the study of nucleic acids in solution.

Project description:Atomic-resolution information on the structure and dynamics of nucleic acids is essential for a better understanding of the mechanistic basis of many cellular processes. NMR spectroscopy is a powerful method for studying the structure and dynamics of nucleic acids; however, solution NMR studies are currently limited to relatively small nucleic acids at high concentrations. Thus, technological and methodological improvements that increase the experimental sensitivity and spectral resolution of NMR spectroscopy are required for studies of larger nucleic acids or protein-nucleic acid complexes. Here we introduce a series of imino-proton-detected NMR experiments that yield an over 2-fold increase in sensitivity compared to conventional pulse schemes. These methods can be applied to the detection of base pair interactions, RNA-ligand titration experiments, measurement of residual dipolar (15)N-(1)H couplings, and direct measurements of conformational transitions. These NMR experiments employ longitudinal spin relaxation enhancement techniques that have proven useful in protein NMR spectroscopy. The performance of these new experiments is demonstrated for a 10 kDa TAR-TAR*(GA) RNA kissing complex and a 26 kDa tRNA.

| S-EPMC2846706 | biostudies-literature

Structures of invisible, excited protein states by relaxation dispersion NMR spectroscopy.

Project description:Molecular function is often predicated on excursions between ground states and higher energy conformers that can play important roles in ligand binding, molecular recognition, enzyme catalysis, and protein folding. The tools of structural biology enable a detailed characterization of ground state structure and dynamics; however, studies of excited state conformations are more difficult because they are of low population and may exist only transiently. Here we describe an approach based on relaxation dispersion NMR spectroscopy in which structures of invisible, excited states are obtained from chemical shifts and residual anisotropic magnetic interactions. To establish the utility of the approach, we studied an exchanging protein (Abp1p SH3 domain)-ligand (Ark1p peptide) system, in which the peptide is added in only small amounts so that the ligand-bound form is invisible. From a collection of (15)N, (1)HN, (13)C(alpha), and (13)CO chemical shifts, along with (1)HN-(15)N, (1)H(alpha)-(13)C(alpha), and (1)HN-(13)CO residual dipolar couplings and (13)CO residual chemical shift anisotropies, all pertaining to the invisible, bound conformer, the structure of the bound state is determined. The structure so obtained is cross-validated by comparison with (1)HN-(15)N residual dipolar couplings recorded in a second alignment medium. The methodology described opens up the possibility for detailed structural studies of invisible protein conformers at a level of detail that has heretofore been restricted to applications involving visible ground states of proteins.

| S-EPMC2575287 | biostudies-literature

Measuring 1HN temperature coefficients in invisible protein states by relaxation dispersion NMR spectroscopy.

Project description:A method based on the Carr-Purcell-Meiboom-Gill relaxation dispersion experiment is presented for measuring the temperature coefficients of amide proton chemical shifts of low populated 'invisible' protein states that exchange with a 'visible' ground state on the millisecond time-scale. The utility of the approach is demonstrated with an application to an I58D mutant of the Pfl6 Cro protein that undergoes exchange between the native, folded state and a cold denatured, unfolded conformational ensemble that is populated at a level of 6% at 2.5°C. A wide distribution of amide temperature coefficients is measured for the unfolded state. The distribution is centered about -5.6 ppb/K, consistent with an absence of intra-molecular hydrogen bonds, on average. However, the large range of values (standard deviation of 2.1 ppb/K) strongly supports the notion that the unfolded state of the protein is not a true random coil polypeptide chain.

| S-EPMC3229278 | biostudies-literature

Transient On- and Off-Pathway Protein Folding Intermediate States Characterized with NMR Relaxation Dispersion.

Project description:The earliest events in the folding of a protein are in general poorly understood. We used NMR R2 relaxation dispersion experiments to study transient local collapse events in the unfolded-state (U) conformational ensemble of apomyoglobin (apoMb). Local residual secondary structure (seen in regions corresponding to the A, D, E, and H helices of the folded protein) is largely unchanged over the pH range of 2.3-2.75, yet a significant pH-dependent increase in the conformational exchange contribution to the R2 relaxation rate (Rex) indicates that transient intramolecular contacts occur on a microsecond to millisecond time scale at pH 2.75. A comparison of 15N and 13CO relaxation dispersion data at pH 2.75 for residues in the A, B, G, and H regions, which participate in the earliest folding intermediates, indicates that chain collapse and secondary structure formation are rapid and concomitant. Increasingly stabilizing conditions (lower temperature, higher pH) result in the observation of a relaxation dispersion in the C, CD, and E regions of the protein, which are known to fold at later stages. Mutation of Trp14 in the A-helix region to Ala eliminates conformational exchange throughout the protein, and the mutation of hydrophobic residues in other regions results in the selective inhibition of conformational exchange in the B, G, or H regions. The R2 dispersion data for WT apoMb at pH 2.75 and 10 °C are best fit to a four-state model ABGH ⇆ AGH ⇆ U ⇆ ABCD that includes on-pathway (AGH and ABGH) and off-pathway (ABCD) transiently folded states, both of which are required to explain the behavior of the mutant proteins. The off-pathway intermediate is destabilized at higher temperatures. Our analysis provides insights into the earliest stages of apoMb folding where the collapsing polypeptide chain samples both productive and nonproductive states with stabilized secondary structure.

| S-EPMC9793904 | biostudies-literature

Mechanism of E-cadherin dimerization probed by NMR relaxation dispersion.

Project description:Epithelial cadherin (E-cadherin), a member of the classical cadherin family, mediates calcium-dependent homophilic cell-cell adhesion. Crystal structures of classical cadherins reveal an adhesive dimer interface featuring reciprocal exchange of N-terminal ?-strands between two protomers. Previous work has identified a putative intermediate (called the "X-dimer") in the dimerization pathway of wild-type E-cadherin EC1-EC2 domains, based on crystal structures of mutants not capable of strand swapping and on deceleration of binding kinetics by mutations at the X-dimer interface. In the present work, NMR relaxation dispersion spectroscopy is used to directly observe and characterize intermediate states without the need to disrupt the strand-swapped binding interface by mutagenesis. The results indicate that E-cadherin forms strand-swapped dimers predominantly by a mechanism in which formation of a weak and short-lived X-dimer-like state precedes the conformational changes required for formation of the mature strand-swapped dimeric structure. Disruption of this intermediate state through mutation reduces both association and dissociation rates by factors of ~10(4), while minimally perturbing affinity. The X-dimer interface lowers the energy barrier associated with strand swapping and enables E-cadherins to form strand-swapped dimers at a rate consistent with residence times in adherens junctions.

| S-EPMC3799306 | 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