Browse
Submit Data
Databases
API
Help

Dataset Information

27 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Specific interaction between GroEL and denatured protein measured by compression-free force spectroscopy.

ABSTRACT: We investigated the interaction between GroEL and a denatured protein from a mechanical point of view using an atomic force microscope. Pepsin was bound to an atomic force microscope probe and used at a neutral pH as an example of denatured proteins. To measure a specific and delicate interaction force, we obtained force curves without pressing the probe onto GroEL molecules spread on a mica surface. Approximately 40 pN of tensile force was observed for approximately 10 nm while pepsin was pulled away from the chaperonin after a brief contact. This length of force duration corresponding to the circumference of GroEL's interior cavity was shortened by the addition of ATP. The relation between the observed mechanical parameters and the chaperonin's refolding function is discussed.

SUBMITTER: Sekiguchi H

PROVIDER: S-EPMC1303104 | biostudies-literature | 2003 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Specific interaction between GroEL and denatured protein measured by compression-free force spectroscopy.

Sekiguchi Hiroshi H Arakawa Hideo H Taguchi Hideki H Ito Takeshi T Kokawa Ryohei R Ikai Atsushi A

Biophysical journal 20030701 1

We investigated the interaction between GroEL and a denatured protein from a mechanical point of view using an atomic force microscope. Pepsin was bound to an atomic force microscope probe and used at a neutral pH as an example of denatured proteins. To measure a specific and delicate interaction force, we obtained force curves without pressing the probe onto GroEL molecules spread on a mica surface. Approximately 40 pN of tensile force was observed for approximately 10 nm while pepsin was pulle ...[more]

PMID: 12829503

Similar Datasets

Correlation between desorption force measured by atomic force microscopy and adsorption free energy measured by surface plasmon resonance spectroscopy for peptide-surface interactions.

Project description:Surface plasmon resonance (SPR) spectroscopy is a useful technique for thermodynamically characterizing peptide-surface interactions; however, its usefulness is limited to the types of surfaces that can readily be formed as thin layers on the nanometer scale on metallic biosensor substrates. Atomic force microscopy (AFM), on the other hand, can be used with any microscopically flat surface, thus making it more versatile for studying peptide-surface interactions. AFM, however, has the drawback of data interpretation due to questions regarding peptide-to-probe-tip density. This problem could be overcome if results from a standardized AFM method could be correlated with SPR results for a similar set of peptide-surface interactions so that AFM studies using the standardized method could be extended to characterize peptide-surface interactions for surfaces that are not amenable for characterization by SPR. In this article, we present the development and application of an AFM method to measure adsorption forces for host-guest peptides sequence on surfaces consisting of alkanethiol self-assembled monolayers (SAMs) with different functionality. The results from these studies show that a linear correlation exists between these data and the adsorption free energy (?G(o)(ads)) values associated with a similar set of peptide-surface systems available from SPR measurements. These methods will be extremely useful to characterize thermodynamically the adsorption behavior for peptides on a much broader range of surfaces than can be used with SPR to provide information related to understanding protein adsorption behavior to these surfaces and to provide an experimental database that can be used for the evaluation, modification, and validation of force field parameters that are needed to represent protein adsorption behavior accurately for molecular simulations.

| S-EPMC3006061 | biostudies-literature

Free-energy changes of bacteriorhodopsin point mutants measured by single-molecule force spectroscopy.

Project description:Single amino acid mutations provide quantitative insight into the energetics that underlie the dynamics and folding of membrane proteins. Chemical denaturation is the most widely used assay and yields the change in unfolding free energy (ΔΔG). It has been applied to >80 different residues of bacteriorhodopsin (bR), a model membrane protein. However, such experiments have several key limitations: 1) a nonnative lipid environment, 2) a denatured state with significant secondary structure, 3) error introduced by extrapolation to zero denaturant, and 4) the requirement of globally reversible refolding. We overcame these limitations by reversibly unfolding local regions of an individual protein with mechanical force using an atomic-force-microscope assay optimized for 2 μs time resolution and 1 pN force stability. In this assay, bR was unfolded from its native bilayer into a well-defined, stretched state. To measure ΔΔG, we introduced two alanine point mutations into an 8-amino-acid region at the C-terminal end of bR's G helix. For each, we reversibly unfolded and refolded this region hundreds of times while the rest of the protein remained folded. Our single-molecule-derived ΔΔG for mutant L223A (-2.3 ± 0.6 kcal/mol) quantitatively agreed with past chemical denaturation results while our ΔΔG for mutant V217A was 2.2-fold larger (-2.4 ± 0.6 kcal/mol). We attribute the latter result, in part, to contact between Val217 and a natively bound squalene lipid, highlighting the contribution of membrane protein-lipid contacts not present in chemical denaturation assays. More generally, we established a platform for determining ΔΔG for a fully folded membrane protein embedded in its native bilayer.

| S-EPMC8020790 | biostudies-literature

Force dependency of biochemical reactions measured by single-molecule force-clamp spectroscopy.

Project description:Here we describe a protocol for using force-clamp spectroscopy to precisely quantify the effect of force on biochemical reactions. A calibrated force is used to control the exposure of reactive sites in a single polyprotein substrate composed of repeated domains. The use of polyproteins allows the identification of successful single-molecule recordings from unambiguous mechanical unfolding fingerprints. Biochemical reactions are then measured directly by detecting the length changes of the substrate held at a constant force. We present the layout of a force-clamp spectrometer along with protocols to design and conduct experiments. These experiments measure reaction kinetics as a function of applied force. We show sample data of the force dependency of two different reactions, protein unfolding and disulfide reduction. These data, which can be acquired in just a few days, reveal mechanistic details of the reactions that currently cannot be resolved by any other technique.

| S-EPMC4676941 | biostudies-literature

Quantifying Instrumental Artifacts in Folding Kinetics Measured by Single-Molecule Force Spectroscopy.

Project description:Force spectroscopy is commonly used to measure the kinetics of processes occurring in single biological molecules. These measurements involve attaching the molecule of interest to micron-sized or larger force probes via compliant linkers. Recent theoretical work has described how the properties of the probes and linkers can alter the observed kinetics from the intrinsic behavior of the molecule in isolation. We applied this theory to estimate the errors in measurements of folding made using optical tweezers. Errors in the folding rates arising from instrument artifacts were only ?20% for constant-force measurements of DNA hairpins with typical choices of linker length and probe size. Measurements of transition paths using a constant trap position at high trap stiffness were also found to be in the low-artifact limit. These results indicate that typical optical trap measurements of kinetics reflect the dynamics of the molecule fairly well, and suggest practical limitations on experimental design to ensure reliable kinetic measurements.

| S-EPMC4968418 | biostudies-literature

Ultra-fast force-clamp spectroscopy data on the interaction between skeletal muscle myosin and actin.

Project description:Ultrafast force-clamp spectroscopy is a single molecule technique based on laser tweezers with sub-millisecond and sub-nanometer resolution. The technique has been successfully applied to investigate the rapid conformational changes that occur when a myosin II motor from skeletal muscle interacts with an actin filament. Here, we share data on the kinetics of such interaction and experimental records collected under different forces [1]. The data can be valuable for researchers interested in the mechanosensitive properties of myosin II, both from an experimental and modeling point of view. The data is related to the research article "ultrafast force-clamp spectroscopy of single molecules reveals load dependence of myosin working stroke" [2].

| S-EPMC6565606 | biostudies-literature

Molten globule-like transition state of protein barnase measured with calorimetric force spectroscopy.

Project description:SignificanceUnderstanding the molecular forces driving the unfolded polypeptide chain to self-assemble into a functional native structure remains an open question. However, identifying the states visited during protein folding (e.g., the transition state between the unfolded and native states) is tricky due to their transient nature. Here, we introduce calorimetric force spectroscopy in a temperature jump optical trap to determine the enthalpy, entropy, and heat capacity of the transition state of protein barnase. We find that the transition state has the properties of a dry molten globule, that is, high free energy and low configurational entropy, being structurally similar to the native state. This experimental single-molecule study characterizes the thermodynamic properties of the transition state in funneled energy landscapes.

| S-EPMC8931224 | biostudies-literature

Substrate protein dependence of GroEL-GroES interaction cycle revealed by high-speed atomic force microscopy imaging.

Project description:A double-ring-shaped tetradecameric GroEL complex assists proper protein folding in cooperation with the cochaperonin GroES. The dynamic GroEL-GroES interaction reflects the allosteric intra- and inter-ring communications and the chaperonin reaction. Therefore, revealing this dynamic interaction is essential to understanding the allosteric communications and the operation mechanism of GroEL. Nevertheless, how this interaction proceeds in the chaperonin cycle has long been controversial. Here, we directly image the dynamic GroEL-GroES interaction under conditions with and without foldable substrate protein using high-speed atomic force microscopy. Then, the imaging results obtained under these conditions and our previous results in the presence of unfoldable substrate are compared. The molecular movies reveal that the entire reaction pathway is highly complicated but basically identical irrespective of the substrate condition. A prominent (but moderate) difference is in the population distribution of intermediate species: symmetric GroEL : GroES2 and asymmetric GroEL : GroES1 complexes, and GroES-unbound GroEL. This difference is mainly attributed to the longer lifetime of GroEL : GroES1 complexes in the presence of foldable substrate. Moreover, the inter-ring communication, which is the basis for the alternating action of the two rings, occurs at two distinct (GroES association and dissociation) steps in the main reaction pathway, irrespective of the substrate condition.This article is part of a discussion meeting issue 'Allostery and molecular machines'.

| S-EPMC5941175 | biostudies-literature

Immune synapse formation determines interaction forces between T cells and antigen-presenting cells measured by atomic force microscopy.

Project description:During adaptive immune responses, T lymphocytes recognize antigenic peptides presented by MHC molecules on antigen-presenting cells (APCs). This recognition results in the formation of a so-called immune synapse (IS) at the T-cell/APC interface, which is crucial for T-cell activation. The molecular composition of the IS has been extensively studied, but little is known about the biophysics and interaction forces between T cells and APCs. Here, we report the measurement of interaction forces between T cells and APCs employing atomic force microscopy (AFM). For these investigations, specific T cells were selected that recognize an antigenic peptide presented by MHC-class II molecules on APCs. Dynamic analysis of T-cell/APC interaction by AFM revealed that in the presence of antigen interaction forces increased from 1 to 2 nN at early time-points to a maximum of approximately 14 nN after 30 min and decreased again after 60 min. These data correlate with the kinetics of synapse formation that also reached a maximum after 30 min, as determined by high-throughput multispectral imaging flow cytometry. Because the integrin lymphocyte function antigen-1 (LFA-1) and its counterpart intercellular adhesion molecule-1 (ICAM-1) are prominent members of a mature IS, the effect of a small molecular inhibitor for LFA-1, BIRT377, was investigated. BIRT377 almost completely abolish the interaction forces, emphasizing the importance of LFA-1/ICAM-1-interactions for firm T-cell/APC adhesion. In conclusion, using biophysical measurements, this study provides precise values for the interaction forces between T cells and APCs and demonstrates that these forces develop over time and are highest when synapse formation is maximal.

| S-EPMC2764924 | biostudies-literature

Investigation of the heparin-thrombin interaction by dynamic force spectroscopy.

Project description:The interaction between heparin and thrombin is a vital step in the blood (anti)coagulation process. Unraveling the molecular basis of the interactions is therefore extremely important in understanding the mechanisms of this complex biological process.In this study, we use a combination of an efficient thiolation chemistry of heparin, a self-assembled monolayer-based single molecule platform, and a dynamic force spectroscopy to provide new insights into the heparin-thrombin interaction from an energy viewpoint at the molecular scale.Well-separated single molecules of heparin covalently attached to mixed self-assembled monolayers are demonstrated, whereby interaction forces with thrombin can be measured via atomic force microscopy-based spectroscopy. Further these interactions are studied at different loading rates and salt concentrations to directly obtain kinetic parameters.An increase in the loading rate shows a higher interaction force between the heparin and thrombin, which can be directly linked to the kinetic dissociation rate constant (koff). The stability of the heparin/thrombin complex decreased with increasing NaCl concentration such that the off-rate was found to be driven primarily by non-ionic forces.These results contribute to understanding the role of specific and nonspecific forces that drive heparin-thrombin interactions under applied force or flow conditions.

| S-EPMC4398634 | biostudies-literature

Comparison of Syringe Compression Force Between ENFit and Legacy Feeding Tubes.

Project description:BACKGROUND:Since initial report in 1972, misconnections continue to be an issue, especially in hospitalized patients with multiple access devices. A new small-bore connector standard (ENFit) has been proposed in order to minimize misconnections. METHODS:Commercially available finalized ENFit tubes of a variety of sizes (14 French [Fr] size, 18Fr, 20Fr, 24Fr, and low-profile) were obtained for current testing. Variety of commercially available formulas including blenderized tube feeding (BTF) products (Jevity 1 Cal, Abbott Nutrition, Abbott Park, IL; Nourish, Functional Formularies, Centerville, OH; Real Food Blends, Chesterton, IN) were tested. RESULTS:Data from individual measurements were aggregated for ENFit and legacy tubes and revealed higher syringe compression force in legacy tubes compared with ENFit tubes for 20Fr size with Jevity formula. Our institution's BTF formula revealed that legacy tubes had lower syringe compression force than ENFit tubes for 14Fr tube size. Remaining measurements revealed no significant difference. Model 1 of regression analysis revealed that only formula and tube size were significant with R2 of 0.63. Model 2 evaluating the impact of tube size, blender, time of blending, and legacy vs ENFit revealed that tube size, blender used, and blending time were significant with legacy vs ENFit being nonsignificant (R2 of 0.72). CONCLUSIONS:Overall, only a small number of tube sizes (14Fr and 20Fr) with selected formulas revealed a significant difference between ENFit and Legacy tubes, with remaining studies finding no significant difference. Regression analysis revealed that variables such as formula, size of tube, blender used, and time of blending may have more impact on compression force.

| S-EPMC6269233 | 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