Browse
Submit Data
Databases
API
Help

Dataset Information

20 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Out-of-Equilibrium Biophysical Chemistry: The Case for Multidimensional, Integrated Single-Molecule Approaches.

ABSTRACT: Out-of-equilibrium processes are ubiquitous across living organisms and all structural hierarchies of life. At the molecular scale, out-of-equilibrium processes (for example, enzyme catalysis, gene regulation, and motor protein functions) cause biological macromolecules to sample an ensemble of conformations over a wide range of time scales. Quantifying and conceptualizing the structure-dynamics to function relationship is challenging because continuously evolving multidimensional energy landscapes are necessary to describe nonequilibrium biological processes in biological macromolecules. In this perspective, we explore the challenges associated with state-of-the-art experimental techniques to understanding biological macromolecular function. We argue that it is time to revisit how we probe and model functional out-of-equilibrium biomolecular dynamics. We suggest that developing integrated single-molecule multiparametric force-fluorescence instruments and using advanced molecular dynamics simulations to study out-of-equilibrium biomolecules will provide a path towards understanding the principles of and mechanisms behind the structure-dynamics to function paradigm in biological macromolecules.

SUBMITTER: Kolimi N

PROVIDER: S-EPMC8474109 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Single-Molecule Analysis beyond Dwell Times: Demonstration and Assessment in and out of Equilibrium.

Project description:We present a simple and robust technique for extracting kinetic rate models and thermodynamic quantities from single-molecule time traces. Single-molecule analysis of complex kinetic sequences (SMACKS) is a maximum-likelihood approach that resolves all statistically relevant rates and also their uncertainties. This is achieved by optimizing one global kinetic model based on the complete data set while allowing for experimental variations between individual trajectories. In contrast to dwell-time analysis, which is the current standard method, SMACKS includes every experimental data point, not only dwell times. As a result, it works as well for long trajectories as for an equivalent set of short ones. In addition, the previous systematic overestimation of fast over slow rates is solved. We demonstrate the power of SMACKS on the kinetics of the multidomain protein Hsp90 measured by single-molecule Förster resonance energy transfer. Experiments in and out of equilibrium are analyzed and compared to simulations, shedding new light on the role of Hsp90's ATPase function. SMACKS resolves accurate rate models even if states cause indistinguishable signals. Thereby, it pushes the boundaries of single-molecule kinetics beyond those of current methods.

| S-EPMC5052450 | biostudies-literature

Conformational equilibrium in supramolecular chemistry: Dibutyltriuret case.

Project description:The association of substituted benzoates and naphthyridine dianions was used to study the complexation of dibutyltriuret. The title molecule is the simplest molecule able to form two intramolecular hydrogen bonds. The naphthyridine salt was used to break two intramolecular hydrogen bonds at a time while with the use of substituted benzoates the systematic approach to study association was achieved. Both, titrations and variable temperature measurements shed the light on the importance of conformational equilibrium and its influence on association in solution. Moreover, the associates were observed by mass spectrometry. The DFT-based computations for complexes and single bond rotational barriers supports experimental data and helps understanding the properties of multiply hydrogen bonded complexes.

| S-EPMC4660960 | biostudies-literature

Enzyme-mediated dynamic combinatorial chemistry allows out-of-equilibrium template-directed synthesis of macrocyclic oligosaccharides.

Project description:We show that the outcome of enzymatic reactions can be manipulated and controlled by using artificial template molecules to direct the self-assembly of specific products in an enzyme-mediated dynamic system. Specifically, we utilize a glycosyltransferase to generate a complex dynamic mixture of interconverting linear and macrocyclic α-1,4-d-glucans (cyclodextrins). We find that the native cyclodextrins (α, β and γ) are formed out-of-equilibrium as part of a kinetically trapped subsystem, that surprisingly operates transiently like a Dynamic Combinatorial Library (DCL) under thermodynamic control. By addition of different templates, we can promote the synthesis of each of the native cyclodextrins with 89-99% selectivity, or alternatively, we can amplify the synthesis of unusual large-ring cyclodextrins (δ and ε) with 9 and 10 glucose units per macrocycle. In the absence of templates, the transient DCL lasts less than a day, and cyclodextrins convert rapidly to short maltooligosaccharides. Templates stabilize the kinetically trapped subsystem enabling robust selective synthesis of cyclodextrins, as demonstrated by the high-yielding sequential interconversion of cyclodextrins in a single reaction vessel. Our results show that given the right balance between thermodynamic and kinetic control, templates can direct out-of-equilibrium self-assembly, and be used to manipulate enzymatic transformations to favor specific and/or alternative products to those selected in Nature.

| S-EPMC6979337 | biostudies-literature

Semisynthetic protein nanoreactor for single-molecule chemistry.

Project description:The covalent chemistry of individual reactants bound within a protein pore can be monitored by observing the ionic current flow through the pore, which acts as a nanoreactor responding to bond-making and bond-breaking events. In the present work, we incorporated an unnatural amino acid into the ?-hemolysin (?HL) pore by using solid-phase peptide synthesis to make the central segment of the polypeptide chain, which forms the transmembrane ?-barrel of the assembled heptamer. The full-length ?HL monomer was obtained by native chemical ligation of the central synthetic peptide to flanking recombinant polypeptides. ?HL pores with one semisynthetic subunit were then used as nanoreactors for single-molecule chemistry. By introducing an amino acid with a terminal alkyne group, we were able to visualize click chemistry at the single-molecule level, which revealed a long-lived (4.5-s) reaction intermediate. Additional side chains might be introduced in a similar fashion, thereby greatly expanding the range of single-molecule covalent chemistry that can be investigated by the nanoreactor approach.

| S-EPMC4653139 | biostudies-literature

Single-Molecule Reaction Chemistry in Patterned Nanowells.

Project description:A new approach to synthetic chemistry is performed in ultraminiaturized, nanofabricated reaction chambers. Using lithographically defined nanowells, we achieve single-point covalent chemistry on hundreds of individual carbon nanotube transistors, providing robust statistics and unprecedented spatial resolution in adduct position. Each device acts as a sensor to detect, in real-time and through quantized changes in conductance, single-point functionalization of the nanotube as well as consecutive chemical reactions, molecular interactions, and molecular conformational changes occurring on the resulting single-molecule probe. In particular, we use a set of sequential bioconjugation reactions to tether a single-strand of DNA to the device and record its repeated, reversible folding into a G-quadruplex structure. The stable covalent tether allows us to measure the same molecule in different solutions, revealing the characteristic increased stability of the G-quadruplex structure in the presence of potassium ions (K(+)) versus sodium ions (Na(+)). Nanowell-confined reaction chemistry on carbon nanotube devices offers a versatile method to isolate and monitor individual molecules during successive chemical reactions over an extended period of time.

| S-EPMC5176326 | biostudies-literature

Physical Chemistry of Epigenetics: Single-Molecule Investigations.

Project description:The nucleosome is the fundamental building block of the eukaryotic genome, composed of an ∼147 base-pair DNA fragment wrapping around an octameric histone protein core. DNA and histone proteins are targets of enzymatic chemical modifications that serve as signals for gene regulation. These modifications are often referred to as epigenetic modifications that govern gene activities without altering the DNA sequence. Although the term epigenetics initially required inheritability, it now frequently includes noninherited histone modifications associated with gene regulation. Important epigenetic modifications for healthy cell growth and proliferation include DNA methylation, histone acetylation, methylation, phosphorylation, ubiquitination, and SUMOylation (SUMO = Small Ubiquitin-like Modifier). Our research focuses on the biophysical roles of these modifications in altering the structure and structural dynamics of the nucleosome and their implications in gene regulation mechanisms. As the changes are subtle and complex, we employ various single-molecule fluorescence approaches for their investigations. Our investigations revealed that these modifications induce changes in the structure and structural dynamics of the nucleosome and their thermodynamic and kinetic stabilities. We also suggested the implications of these changes in gene regulation mechanisms that are the foci of our current and future research.

| S-EPMC6790939 | biostudies-literature

DNA Y structure: a versatile, multidimensional single molecule assay.

Project description:Optical trapping is a powerful single molecule technique used to study dynamic biomolecular events, especially those involving DNA and DNA-binding proteins. Current implementations usually involve only one of stretching, unzipping, or twisting DNA along one dimension. To expand the capabilities of optical trapping for more complex measurements would require a multidimensional technique that combines all of these manipulations in a single experiment. Here, we report the development and utilization of such a novel optical trapping assay based on a three-branch DNA construct, termed a "Y structure". This multidimensional assay allows precise, real-time tracking of multiple configurational changes. When the Y structure template is unzipped under both force and torque, the force and extension of all three branches can be determined simultaneously. Moreover, the assay is readily compatible with fluorescence, as demonstrated by unzipping through a fluorescently labeled, paused transcription complex. This novel assay thus allows for the visualization and precision mapping of complex interactions of biomechanical events.

| S-EPMC4245981 | biostudies-literature

Plasmonic tunnel junctions for single-molecule redox chemistry.

Project description:Nanoparticles attached just above a flat metallic surface can trap optical fields in the nanoscale gap. This enables local spectroscopy of a few molecules within each coupled plasmonic hotspot, with near thousand-fold enhancement of the incident fields. As a result of non-radiative relaxation pathways, the plasmons in such sub-nanometre cavities generate hot charge carriers, which can catalyse chemical reactions or induce redox processes in molecules located within the plasmonic hotspots. Here, surface-enhanced Raman spectroscopy allows us to track these hot-electron-induced chemical reduction processes in a series of different aromatic molecules. We demonstrate that by increasing the tunnelling barrier height and the dephasing strength, a transition from coherent to hopping electron transport occurs, enabling observation of redox processes in real time at the single-molecule level.

| S-EPMC5714966 | biostudies-literature

Semisynthetic Nanoreactor for Reversible Single-Molecule Covalent Chemistry.

Project description:Protein engineering has been used to remodel pores for applications in biotechnology. For example, the heptameric ?-hemolysin pore (?HL) has been engineered to form a nanoreactor to study covalent chemistry at the single-molecule level. Previous work has been confined largely to the chemistry of cysteine side chains or, in one instance, to an irreversible reaction of an unnatural amino acid side chain bearing a terminal alkyne. Here, we present four different ?HL pores obtained by coupling either two or three fragments by native chemical ligation (NCL). The synthetic ?HL monomers were folded and incorporated into heptameric pores. The functionality of the pores was validated by hemolysis assays and by single-channel current recording. By using NCL to introduce a ketone amino acid, the nanoreactor approach was extended to an investigation of reversible covalent chemistry on an unnatural side chain at the single-molecule level.

| S-EPMC5043417 | biostudies-literature

Biophysical characterization of DNA binding from single molecule force measurements.

Project description:Single molecule force spectroscopy is a powerful method that uses the mechanical properties of DNA to explore DNA interactions. Here we describe how DNA stretching experiments quantitatively characterize the DNA binding of small molecules and proteins. Small molecules exhibit diverse DNA binding modes, including binding into the major and minor grooves and intercalation between base pairs of double-stranded DNA (dsDNA). Histones bind and package dsDNA, while other nuclear proteins such as high mobility group proteins bind to the backbone and bend dsDNA. Single-stranded DNA (ssDNA) binding proteins slide along dsDNA to locate and stabilize ssDNA during replication. Other proteins exhibit binding to both dsDNA and ssDNA. Nucleic acid chaperone proteins can switch rapidly between dsDNA and ssDNA binding modes, while DNA polymerases bind both forms of DNA with high affinity at distinct binding sites at the replication fork. Single molecule force measurements quantitatively characterize these DNA binding mechanisms, elucidating small molecule interactions and protein function.

| S-EPMC2930095 | 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