Browse
Submit Data
Databases
API
Help

Dataset Information

16 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Bifunctional nanoarrays for probing the immune response at the single-molecule level.

ABSTRACT: Bifunctional nanoarrays were created to simulate the immunological synapse and probe the T-cell immune response at the single-molecule level. Sub-5 nm AuPd nanodot arrays were fabricated using both e-beam and nanoimprint lithography. The nanoarrays were then functionalized by two costimulatory molecules: antibody UCHT1 Fab, which binds to the T-cell receptor (TCR) and activates the immune response, bound to metallic nanodots; and intercellular adhesion molecule-1, which enhances cell adhesion, on the surrounding area. Initial T-cell experiments show successful attachment and activation on the bifunctional nanoarrays. This nanoscale platform for single-molecule control of TCR in living T-cells provides a new approach to explore how its geometric arrangement affects T-cell activation and behavior, with potential applications in immunotherapy. This platform also serves as a general model for single-molecule nanoarrays where more than one molecular species is required.

SUBMITTER: Cai H

PROVIDER: S-EPMC3808416 | biostudies-other | 2013 Nov

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Probing transcription factor dynamics at the single-molecule level in a living cell.

Project description:Transcription factors regulate gene expression through their binding to DNA. In a living Escherichia coli cell, we directly observed specific binding of a lac repressor, labeled with a fluorescent protein, to a chromosomal lac operator. Using single-molecule detection techniques, we measured the kinetics of binding and dissociation of the repressor in response to metabolic signals. Furthermore, we characterized the nonspecific binding to DNA, one-dimensional (1D) diffusion along DNA segments, and 3D translocation among segments through cytoplasm at the single-molecule level. In searching for the operator, a lac repressor spends approximately 90% of time nonspecifically bound to and diffusing along DNA with a residence time of <5 milliseconds. The methods and findings can be generalized to other nucleic acid binding proteins.

| S-EPMC2853898 | biostudies-literature

Single-molecule correlated chemical probing of RNA.

Project description:Complex higher-order RNA structures play critical roles in all facets of gene expression; however, the through-space interaction networks that define tertiary structures and govern sampling of multiple conformations are poorly understood. Here we describe single-molecule RNA structure analysis in which multiple sites of chemical modification are identified in single RNA strands by massively parallel sequencing and then analyzed for correlated and clustered interactions. The strategy thus identifies RNA interaction groups by mutational profiling (RING-MaP) and makes possible two expansive applications. First, we identify through-space interactions, create 3D models for RNAs spanning 80-265 nucleotides, and characterize broad classes of intramolecular interactions that stabilize RNA. Second, we distinguish distinct conformations in solution ensembles and reveal previously undetected hidden states and large-scale structural reconfigurations that occur in unfolded RNAs relative to native states. RING-MaP single-molecule nucleic acid structure interrogation enables concise and facile analysis of the global architectures and multiple conformations that govern function in RNA.

| S-EPMC4183288 | biostudies-literature

Probing DNA clamps with single-molecule force spectroscopy.

Project description:Detailed mechanisms of DNA clamps in prokaryotic and eukaryotic systems were investigated by probing their mechanics with single-molecule force spectroscopy. Specifically, the mechanical forces required for the Escherichia coli and Saccharomyces cerevisiae clamp opening were measured at the single-molecule level by optical tweezers. Steered molecular dynamics simulations further examined the forces involved in DNA clamp opening from the perspective of the interface binding energies associated with the clamp opening processes. In combination with additional molecular dynamics simulations, we identified the contact networks between the clamp subunits that contribute significantly to the interface stability of the S.cerevisiae and E. coli clamps. These studies provide a vivid picture of the mechanics and energy landscape of clamp opening and reveal how the prokaryotic and eukaryotic clamps function through different mechanisms.

| S-EPMC3763527 | biostudies-literature

Probing cellular protein complexes using single-molecule pull-down.

Project description:Proteins perform most cellular functions in macromolecular complexes. The same protein often participates in different complexes to exhibit diverse functionality. Current ensemble approaches of identifying cellular protein interactions cannot reveal physiological permutations of these interactions. Here we describe a single-molecule pull-down (SiMPull) assay that combines the principles of a conventional pull-down assay with single-molecule fluorescence microscopy and enables direct visualization of individual cellular protein complexes. SiMPull can reveal how many proteins and of which kinds are present in the in vivo complex, as we show using protein kinase A. We then demonstrate a wide applicability to various signalling proteins found in the cytosol, membrane and cellular organelles, and to endogenous protein complexes from animal tissue extracts. The pulled-down proteins are functional and are used, without further processing, for single-molecule biochemical studies. SiMPull should provide a rapid, sensitive and robust platform for analysing protein assemblies in biological pathways.

| S-EPMC3103084 | biostudies-literature

Time-Resolved, Single-Molecule, Correlated Chemical Probing of RNA.

Project description:Capturing the folding dynamics of large, functionally important RNAs has relied primarily on global measurements of structure or on per-nucleotide chemical probing. These approaches infer, but do not directly measure, through-space structural interactions. Here we introduce trimethyloxonium (TMO) as a chemical probe for RNA. TMO alkylates RNA at high levels in seconds, and thereby enables time-resolved, single-molecule, through-space probing of RNA folding using the RING-MaP correlated chemical probing framework. Time-resolved correlations in the RNase P RNA-a functional RNA with a complex structure stabilized by multiple noncanonical interactions-revealed that a long-range tertiary interaction guides native RNA folding for both secondary and tertiary structure. This unanticipated nonhierarchical folding mechanism was directly validated by examining the consequences of concise disruption of the through-space interaction. Single-molecule, time-resolved RNA structure probing with TMO is poised to reveal a wide range of dynamic RNA folding processes and principles of RNA folding.

| S-EPMC7917579 | biostudies-literature

Probing single-molecule enzyme active-site conformational state intermittent coherence.

Project description:The relationship between protein conformational dynamics and enzymatic reactions has been a fundamental focus in modern enzymology. Using single-molecule fluorescence resonance energy transfer (FRET) with a combined statistical data analysis approach, we have identified the intermittently appearing coherence of the enzymatic conformational state from the recorded single-molecule intensity-time trajectories of enzyme 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) in catalytic reaction. The coherent conformational state dynamics suggests that the enzymatic catalysis involves a multistep conformational motion along the coordinates of substrate-enzyme complex formation and product releasing, presenting as an extreme dynamic behavior intrinsically related to the time bunching effect that we have reported previously. The coherence frequency, identified by statistical results of the correlation function analysis from single-molecule FRET trajectories, increases with the increasing substrate concentrations. The intermittent coherence in conformational state changes at the enzymatic reaction active site is likely to be common and exist in other conformation regulated enzymatic reactions. Our results of HPPK interaction with substrate support a multiple-conformational state model, being consistent with a complementary conformation selection and induced-fit enzymatic loop-gated conformational change mechanism in substrate-enzyme active complex formation.

| S-EPMC3198842 | biostudies-literature

Single Molecule Probing of Exocytotic Protein Interactions Using Force Spectroscopy.

Project description:Relatively recently, the Atomic Force Microscope (AFM) emerged as a powerful tool for single molecule nanomechanical investigations. Parameters that can be measured by force spectroscopy using AFM, such as the force and total mechanical extension required to break bonds between various proteins can yield valuable insights into the nature of the bond (zippering vs. highly localized binding site), the sequence of its interactions and the energy landscape along the length of the interaction. In this review we discuss the use of AFM in force spectroscopy mode to study intermolecular interactions between the exocytotic proteins of the core SNARE complex. Information gathered by force spectroscopy of protein-protein interactions of this complex supplement previous results acquired with other techniques, and allows a deeper understanding of SNARE protein interactions and their role in exocytosis.

| S-EPMC2819743 | biostudies-literature

Imaging proteins at the single-molecule level.

Project description:Imaging single proteins has been a long-standing ambition for advancing various fields in natural science, as for instance structural biology, biophysics, and molecular nanotechnology. In particular, revealing the distinct conformations of an individual protein is of utmost importance. Here, we show the imaging of individual proteins and protein complexes by low-energy electron holography. Samples of individual proteins and protein complexes on ultraclean freestanding graphene were prepared by soft-landing electrospray ion beam deposition, which allows chemical- and conformational-specific selection and gentle deposition. Low-energy electrons do not induce radiation damage, which enables acquiring subnanometer resolution images of individual proteins (cytochrome C and BSA) as well as of protein complexes (hemoglobin), which are not the result of an averaging process.

| S-EPMC5321008 | biostudies-literature

Probing Single Vacancies in Black Phosphorus at the Atomic Level.

Project description:Utilizing a combination of low-temperature scanning tunneling microscopy/spectroscopy (STM/STS) and electronic structure calculations, we characterize the structural and electronic properties of single atomic vacancies within several monolayers of the surface of black phosphorus. We illustrate, with experimental analysis and tight-binding calculations, that we can depth profile these vacancies and assign them to specific sublattices within the unit cell. Measurements reveal that the single vacancies exhibit strongly anisotropic and highly delocalized charge density, laterally extended up to 20 atomic unit cells. The vacancies are then studied with STS, which reveals in-gap resonance states near the valence band edge and a strong p-doping of the bulk black phosphorus crystal. Finally, quasiparticle interference generated near these vacancies enables the direct visualization of the anisotropic band structure of black phosphorus.

| S-EPMC5474690 | biostudies-literature

Probing static disorder in Arrhenius kinetics by single-molecule force spectroscopy.

Project description:The widely used Arrhenius equation describes the kinetics of simple two-state reactions, with the implicit assumption of a single transition state with a well-defined activation energy barrier DeltaE, as the rate-limiting step. However, it has become increasingly clear that the saddle point of the free-energy surface in most reactions is populated by ensembles of conformations, leading to nonexponential kinetics. Here we present a theory that generalizes the Arrhenius equation to include static disorder of conformational degrees of freedom as a function of an external perturbation to fully account for a diverse set of transition states. The effect of a perturbation on static disorder is best examined at the single-molecule level. Here we use force-clamp spectroscopy to study the nonexponential kinetics of single ubiquitin proteins unfolding under force. We find that the measured variance in DeltaE shows both force-dependent and independent components, where the force-dependent component scales with F(2), in excellent agreement with our theory. Our study illustrates a novel adaptation of the classical Arrhenius equation that accounts for the microscopic origins of nonexponential kinetics, which are essential in understanding the rapidly growing body of single-molecule data.

| S-EPMC2895103 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

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