Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Fluorescence quenching by TEMPO: a sub-30 A single-molecule ruler.

ABSTRACT: A series of DNA molecules labeled with 5-carboxytetramethylrhodamine (5-TAMRA) and the small nitroxide radical TEMPO were synthesized and tested to investigate whether the intramolecular quenching efficiency can be used to measure short intramolecular distances in small ensemble and single-molecule experiments. In combination with distance calculations using molecular mechanics modeling, the experimental results from steady-state ensemble fluorescence and fluorescence correlation spectroscopy measurements both show an exponential decrease in the quenching rate constant with the dye-quencher distance in the 10-30 A range. The results demonstrate that TEMPO-5-TAMRA fluorescence quenching is a promising method to measure short distance changes within single biomolecules.

SUBMITTER: Zhu P

PROVIDER: S-EPMC1366861 | biostudies-literature | 2005 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Fluorescence quenching by TEMPO: a sub-30 A single-molecule ruler.

Zhu Peizhi P Clamme Jean-Pierre JP Deniz Ashok A AA

Biophysical journal 20050930 5

A series of DNA molecules labeled with 5-carboxytetramethylrhodamine (5-TAMRA) and the small nitroxide radical TEMPO were synthesized and tested to investigate whether the intramolecular quenching efficiency can be used to measure short intramolecular distances in small ensemble and single-molecule experiments. In combination with distance calculations using molecular mechanics modeling, the experimental results from steady-state ensemble fluorescence and fluorescence correlation spectroscopy me ...[more]

PMID: 16199509

Similar Datasets

Fluorescence quenching: A tool for single-molecule protein-folding study.

Project description:By using titin as a model system, we have demonstrated that fluorescence quenching can be used to study protein folding at the single molecule level. The unfolded titin molecules with multiple dye molecules attached are able to fold to the native state. In the native folded state, the fluorescence from dye molecules is quenched due to the close proximity between the dye molecules. Unfolding of the titin leads to a dramatic increase in the fluorescence intensity. Such a change makes the folded and unfolded states of a single titin molecule clearly distinguishable and allows us to measure the folding dynamics of individual titin molecules in real time. We have also shown that fluorescence quenching can signal folding and unfolding of a small protein with only one immunoglobulin domain.

| S-EPMC18902 | biostudies-literature

Ensemble and single-molecule studies on fluorescence quenching in transition metal bipyridine-complexes.

Project description:Beyond their use in analytical chemistry fluorescent probes continuously gain importance because of recent applications of single-molecule fluorescence spectroscopy to monitor elementary reaction steps. In this context, we characterized quenching of a fluorescent probe by different metal ions with fluorescence spectroscopy in the bulk and at the single-molecule level. We apply a quantitative model to explain deviations from existing standard models for fluorescence quenching. The model is based on a reversible transition from a bright to a dim state upon binding of the metal ion. We use the model to estimate the stability constants of complexes with different metal ions and the change of the relative quantum yield of different reporter dye labels. We found ensemble data to agree widely with results from single-molecule experiments. Our data indicates a mechanism involving close molecular contact of dye and quenching moiety which we also found in molecular dynamics simulations. We close the manuscript with a discussion of possible mechanisms based on Förster distances and electrochemical potentials which renders photo-induced electron transfer to be more likely than Förster resonance energy transfer.

| S-EPMC3587577 | biostudies-literature

Fluorescence Quenching Studies on the Interactions between Chosen Fluoroquinolones and Selected Stable TEMPO and PROXYL Nitroxides.

Project description:The influence of the stable 2,2,6,6-tetramethylpiperidinyl-N-oxyl (TEMPO) nitroxide and its six C4-substituted derivatives, as well as two C3-substituted analogues of 2,2,5,5-tetramethylpyrrolidynyl-N-oxyl (PROXYL) nitroxide on the chosen fluoroquinolone antibiotics (marbofloxacin, ciprofloxacin, danofloxacin, norfloxacin, enrofloxacin, levofloxacin and ofloxacin), has been examined in aqueous solutions by UV absorption as well as steady-state and time-resolved fluorescence spectroscopies. The mechanism of fluorescence quenching has been specified and proved to be purely dynamic (collisional) for all the studied systems, which was additionally confirmed by temperature dependence experiments. Moreover, the selected quenching parameters-that is, Stern-Volmer quenching constants and bimolecular quenching rate constants-have been determined and explained. The possibility of electron transfer was ruled out, and the quenching was found to be diffusion-limited, being a result of the increase in non-radiative processes. Furthermore, as the chosen nitroxides affected the fluorescence of fluoroquinolone antibiotics in different ways, an influence of the structure and the type of substituents in the molecules of both fluoroquinolones and stable radicals on the quenching efficiency has been determined and discussed. Finally, the impact of the solvent's polarity on the values of bimolecular quenching rate constants has been explained. The significance of the project comes from many applications of nitroxides in chemistry, biology and industry.

| S-EPMC7830230 | biostudies-literature

Short-range spectroscopic ruler based on a single-molecule optical switch.

Project description:We demonstrate a novel all-optical switch consisting of two molecules: a primary fluorophore (Cy5) that can be switched between a fluorescent and a dark state by light of different wavelengths, and a secondary chromophore (Cy3) that facilitates switching. The interaction between the two molecules exhibits a distance dependence much steeper than that of conventional Fo rster resonance energy transfer. This enables the switch to act as a ruler with the capability to probe distances difficult to access by other spectroscopic methods, thus presenting a new tool for the study of biomolecules at the single-molecule level.

| S-EPMC2652517 | biostudies-literature

Implementing fluorescence enhancement, quenching, and FRET for investigating flap endonuclease 1 enzymatic reaction at the single-molecule level.

Project description:Flap endonuclease 1 (FEN1) is an important component of the intricate molecular machinery for DNA replication and repair. FEN1 is a structure-specific 5' nuclease that cleaves nascent single-stranded 5' flaps during the maturation of Okazaki fragments. Here, we review our research primarily applying single-molecule fluorescence to resolve important mechanistic aspects of human FEN1 enzymatic reaction. The methodology presented in this review is aimed as a guide for tackling other biomolecular enzymatic reactions by fluorescence enhancement, quenching, and FRET and their combinations. Using these methods, we followed in real-time the structures of the substrate and product and 5' flap cleavage during catalysis. We illustrate that FEN1 actively bends the substrate to verify its features and continues to mold it to induce a protein disorder-to-order transitioning that controls active site assembly. This mechanism suppresses off-target cleavage of non-cognate substrates and promotes their dissociation with an accuracy that was underestimated from bulk assays. We determined that product release in FEN1 after the 5' flap release occurs in two steps; a brief binding to the bent nicked-product followed by longer binding to the unbent nicked-product before dissociation. Based on our cryo-electron microscopy structure of the human lagging strand replicase bound to FEN1, we propose how this two-step product release mechanism may regulate the final steps during the maturation of Okazaki fragments.

| S-EPMC8385120 | biostudies-literature

Quenching of Protein Fluorescence by Fullerenol C60(OH)36 Nanoparticles.

Project description:The effect of the interaction between fullerenol C60(OH)36 (FUL) and alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae and human serum albumin (HSA) was studied by absorption spectroscopy, fluorescence spectroscopy, and time-resolved fluorescence spectroscopy. As shown in the study, the fluorescence intensities of ADH and HSA at excitation wavelengths λex = 280 nm (Trp, Tyr) and λex = 295 nm (Trp) are decreased with the increase in the FUL concentration. The results of time-resolved measurements indicate that both quenching mechanisms, dynamic and static, are present. The binding constant Kb and the number of binding sites were obtained for HSA and ADH. Thus, the results indicated the formation of FUL complexes and proteins. However, the binding of FUL to HSA is much stronger than that of ADH. The transfer of energy from the protein to FUL was also proved.

| S-EPMC9603995 | biostudies-literature

Hydrogen release from a single water molecule on Vn+ (3 ≤ n ≤ 30).

Project description:Water and its interactions with metals are closely bound up with human life, and the reactivity of metal clusters with water is of fundamental importance for the understanding of hydrogen generation. Here a prominent hydrogen evolution reaction (HER) of single water molecule on vanadium clusters Vn+ (3 ≤ n ≤ 30) is observed in the reaction of cationic vanadium clusters with water at room temperature. The combined experimental and theoretical studies reveal that the wagging vibrations of a V-OH group give rise to readily formed V-O-V intermediate states on Vn+ (n ≥ 3) clusters and allow the terminal hydrogen to interact with an adsorbed hydrogen atom, enabling hydrogen release. The presence of three metal atoms reduces the energy barrier of the rate-determining step, giving rise to an effective production of hydrogen from single water molecules. This mechanism differs from dissociative chemisorption of multiple water molecules on aluminium cluster anions, which usually proceeds by dissociative chemisorption of at least two water molecules at multiple surface sites followed by a recombination of the adsorbed hydrogen atoms.

| S-EPMC9814650 | biostudies-literature

Synthetic and genetic dimers as quantification ruler for single-molecule counting with PALM.

Project description:How membrane proteins oligomerize determines their function. Superresolution microscopy can report on protein clustering and extract quantitative molecular information. Here, we evaluate the blinking kinetics of four photoactivatable fluorescent proteins for quantitative single-molecule microscopy. We identified mEos3.2 and mMaple3 to be suitable for molecular quantification through blinking histogram analysis. We designed synthetic and genetic dimers of mEos3.2 as well as fusion proteins of monomeric and dimeric membrane proteins as reference structures, and we demonstrate their versatile use for quantitative superresolution imaging in vitro and in situ. We further found that the blinking behavior of mEos3.2 and mMaple3 is modified by a reducing agent, offering the possibility to adjust blinking parameters according to experimental needs.

| S-EPMC6724688 | biostudies-literature

Amyloidophilic Molecule Interactions on the Surface of Insulin Fibrils: Cooperative Binding and Fluorescence Quenching.

Project description:Protein aggregation into insoluble fibrillar aggregates is linked to several neurodegenerative disorders, such as Alzheimer's or Parkinson's disease. Commonly used methods to study aggregation inhibition or fibril destabilization by potential drugs include spectroscopic measurements of amyloidophilic dye molecule fluorescence or absorbance changes. In this work we show the cross-interactions of five different dye molecules on the surface of insulin amyloid fibrils, resulting in cooperative binding and fluorescence quenching.

| S-EPMC6937241 | biostudies-literature

Method for high frequency tracking and sub-nm sample stabilization in single molecule fluorescence microscopy.

Project description:While fluorescence microscopes and atomic force microscopes are widely used to visualize, track, and manipulate single biomolecules, the resolution of these methods is limited by sample drift. To minimize drift, active feedback methods have recently been used to stabilize single molecule microscopes on the sub-nanometer scale. However, these methods require high intensity lasers which limits their application in single molecule fluorescence measurements. Furthermore, these feedback methods do not track user-defined regions of the sample, but rather monitor the relative displacement of an unknown point on a fiducial marker, which limits their use in biological force measurements. To overcome these limitations, we have developed a novel method to image, track and stabilize a sample using low laser intensities. We demonstrate the capabilities of our approach by tracking a user-chosen point on a fiducial marker at 8.6 kHz and stabilizing it with sub-nanometer resolution. We further showcase the application of our method in single molecule fluorescence microscopy by imaging and stabilizing individual fluorescently-tagged streptavidin proteins under biologically relevant conditions. We anticipate that our method can be easily used to improve the resolution of a wide range of single molecule fluorescence microscopy and integrated force-fluorescence applications.

| S-EPMC6141618 | 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