Browse
Submit Data
Databases
API
Help

Dataset Information

20 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Side-chain thioamides as fluorescence quenching probes.

ABSTRACT: Thioamides, single atom oxygen-to-sulfur substitutions of canonical amide bonds, can be valuable probes for protein folding and protease studies. Here, we investigate the fluorescence quenching properties of thioamides incorporated into the side-chains of amino acids. We synthesize and incorporate Fmoc-protected, solid-phase peptide synthesis building blocks for introducing N^? -thioacetyl-lysine and ?-thioasparagine. Using rigid model peptides, we demonstrate the distance-dependent fluorescence quenching of these thioamides. Furthermore, we describe attempts to incorporate of N^? -thioacetyl-lysine into proteins expressed in Escherichia coli using amber codon suppression.

SUBMITTER: Robkis DM

PROVIDER: S-EPMC7744324 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Side-chain thioamides as fluorescence quenching probes.

Robkis D Miklos DM Hoang Eileen M EM Po Pengse P Deutsch Carol J CJ Petersson E James EJ

Biopolymers 20200617 1

Thioamides, single atom oxygen-to-sulfur substitutions of canonical amide bonds, can be valuable probes for protein folding and protease studies. Here, we investigate the fluorescence quenching properties of thioamides incorporated into the side-chains of amino acids. We synthesize and incorporate Fmoc-protected, solid-phase peptide synthesis building blocks for introducing N<sup>ε</sup> -thioacetyl-lysine and γ-thioasparagine. Using rigid model peptides, we demonstrate the distance-dependent fl ...[more]

PMID: 32740927

Similar Datasets

Investigation of eco-friendly fluorescence quenching probes for assessment of acemetacin using silver nanoparticles and acriflavine reagent.

Project description:The non-steroidal anti-inflammatory medication acemetacin was assessed via two straightforward green spectrofluorimetric techniques. The quenching-dependent derivatizing spectrofluorimetric reactions are the master point of this study. Acriflavine-based method (Method I) depends on forming an ion association complex between acriflavine and the drug in a ratio of 1:1, decreasing the former's fluorescence intensity. Acriflavine or Ag NP's intensity-related quenching action goes linearly with the acemetacin concentration in the 2.0-20.0 µg/mL and 1.0-16.0 µg/mL ranges, respectively. The second quenching mechanism depends on using the silver nanoparticles (Ag NP's) as a fluorescence probe (Method II); Ag NP's were prepared from reducing silver nitrate using sodium borohydride. Both methods could be applied to determine pure and pharmaceutical dosage forms of acemetacin. The methods proved valid according to the international conference on harmonization (ICH) guidelines. In addition to this, this work has been estimated under green criteria assessment tools. There is no significant difference between the proposed and the comparison methods after the statistical interpretation.

| S-EPMC10014932 | biostudies-literature

Steady-state and time-resolved fluorescence quenching with transition metal ions as short-distance probes for protein conformation.

Project description:A series of model dye-labeled histidine-containing peptides was used to investigate the nature of the quenching mechanism with Cu(2+) and Ni(2+). The strong reduction in steady-state fluorescence was found to be unaccompanied by any noticeable changes in lifetime kinetics. This static nature of quenching is not consistent with the dynamic Förster resonance energy transfer (FRET) phenomenon, which was assumed to dominate the quenching mechanism, and is likely caused by shorter range orbital coupling. Our results indicate that the FRET-like sixth power of distance dependence of quenching cannot be automatically assumed for transition metal ions and that time-resolved measurements should be used to distinguish various quenching mechanisms.

| S-EPMC3204958 | biostudies-literature

Modulation of p-cyanophenylalanine fluorescence by amino acid side chains and rational design of fluorescence probes of alpha-helix formation.

Project description:p-Cyanophenylalanine is an extremely useful fluorescence probe of protein structure that can be recombinantly and chemically incorporated into proteins. The probe has been used to study protein folding, protein-membrane interactions, protein-peptide interactions, and amyloid formation; however, the factors that control its fluorescence are not fully understood. Hydrogen bonding to the cyano group is known to play a major role in modulating the fluorescence quantum yield, but the role of potential side-chain quenchers has not yet been elucidated. A systematic study of the effects of different side chains on p-cyanophenylalanine fluorescence is reported. Tyr is found to have the largest effect followed by deprotonated His, Met, Cys, protonated His, Asn, Arg, and protonated Lys. Deprotonated amino groups are much more effective fluorescence quenchers than protonated amino groups. Free neutral imidazole and hydroxide ion are also effective quenchers of p-cyanophenylalanine fluorescence with Stern-Volmer constants of 39.8 and 22.1 M(-1), respectively. The quenching of p-cyanophenylalanine fluorescence by specific side chains is exploited in developing specific, high-sensitivity, fluorescence probes of helix formation. The approach is demonstrated with Ala-based peptides that contain a p-cyanophenylalanine-His or a p-cyanophenylalanine-Tyr pair located at positions i and i + 4. The p-cyanophenylalanine-His pair is most useful when the His side chain is deprotonated and is, thus, complementary to the Trp-His pair which is most sensitive when the His side chain is protonated.

| S-EPMC3199967 | biostudies-literature

DNA analysis by fluorescence quenching detection.

Project description:The analysis of human genetic variations such as single nucleotide polymorphisms (SNPs) has great applications in genome-wide association studies of complex genetic traits. We have developed an SNP genotyping method based on the primer extension assay with fluorescence quenching as the detection. The template-directed dye-terminator incorporation with fluorescence quenching detection (FQ-TDI) assay is based on the observation that the intensity of fluorescent dye R110- and R6G-labeled acycloterminators is universally quenched once they are incorporated onto a DNA oligonucleotide primer. By comparing the rate of fluorescence quenching of the two allelic dyes in real time, we have extended this method for allele frequency estimation of SNPs in pooled DNA samples. The kinetic FQ-TDI assay is highly accurate and reproducible both in genotyping and in allele frequency estimation. Allele frequencies estimated by the kinetic FQ-TDI assay correlated well with known allele frequencies, with an r(2) value of 0.993. Applying this strategy to large-scale studies will greatly reduce the time and cost for genotyping hundreds and thousands of SNP markers between affected and control populations.

| S-EPMC430929 | biostudies-literature

Thioamide quenching of intrinsic protein fluorescence.

Project description:Thioamides quench tryptophan and tyrosine fluorescence in a distance-dependent manner and thus can be used to monitor the binding of thioamide-containing peptides to proteins. Since thioamide analogs of the natural amino acids can be synthetically incorporated into peptides, they can function as minimally-perturbing probes of protein/peptide interactions.

| S-EPMC7759719 | biostudies-literature

Fluorescence determination of tryptophan side-chain accessibility and dynamics in triple-helical collagen-like peptides.

Project description:We report tryptophan fluorescence measurements of emission intensity, iodide quenching, and anisotropy that describe the environment and dynamics at X and Y sites in stable collagen-like peptides of sequence (Gly-X-Y)(n). About 90% of tryptophans at both sites have similar solvent exposed fluorescence properties and a lifetime of 8.5-9 ns. Analysis of anisotropy decays using an associative model indicates that these long lifetime populations undergo rapid depolarizing motion with a 0.5 ns correlation time; however, the extent of fast motion at the Y site is considerably less than the essentially unrestricted motion at the X site. About 10% of tryptophans at both sites have a shorter ( approximately 3 ns) lifetime indicating proximity to a protein quenching group; these minor populations are immobile on the peptide surface, depolarizing only by overall trimer rotation. Iodide quenching indicates that tryptophans at the X site are more accessible to solvent. Side chains at X sites are more solvent accessible and considerably more mobile than residues at Y sites and can more readily fluctuate among alternate intermolecular interactions in collagen fibrils. This fluorescence analysis of collagen-like peptides lays a foundation for studies on the structure, dynamics, and function of collagen and of triple-helical junctions in gelatin gels.

| S-EPMC1302630 | biostudies-literature

Intrinsic Fluorescence of PAMAM Dendrimers-Quenching Studies.

Project description:Intrinsic, non-traditional fluorescence of polyamidoamine (PAMAM) dendrimers that do not possess classical fluorophores has been attracting considerable interest for the last decade. Many hypotheses regarding the source of the fluorescence have appeared, but some of them are still disputable. In order to shed new light on the nature of the phenomenon, we applied quenchers that are normally used to study intrinsic fluorescence of proteins (i.e., KI, CsCl, and acrylamide). KI and acrylamide efficiently quenched steady state fluorescence of PAMAM G2, PAMAM G3, and PAMAM G4 dendrimers. Stern-Volmer plots exhibited a downward curvature that has been elucidated by heterogenous emission. We assume that there are two distinct fluorescent moieties in the dendrimer structure that are characterized by different accessibility to the quenchers.

| S-EPMC6415388 | biostudies-literature

Fluorescence quenching of human orosomucoid. Accessibility to drugs and small quenching agents.

Project description:The fluorescence behaviour of human orosomucoid was investigated. The intrinsic fluorescence was more accessible to acrylamide than to the slightly larger succinimide, indicating limited accessibility to part of the tryptophan population. Although I- showed almost no quenching, that of Cs+ was enhanced, and suggested a region of negative charge proximal to an emitting tryptophan residue. Removal of more than 90% of sialic acid from the glycan chains led to no change in the Cs+, I-, succinimide or acrylamide quenching, indicating that the negatively charged region originates with the protein core. Quenching as a function of pH and temperature supported this view. The binding of chlorpromazine monitored by fluorescence quenching, in the presence and in the absence of the small quenching probes (above), led to a model of its binding domain on orosomucoid that includes two tryptophan residues relatively shielded from the bulk solvent, with the third tryptophan residue being on the periphery of the domain, or affected allotopically and near the negatively charged field.

| S-EPMC1152962 | biostudies-other

Arginine Side-Chain Hydrogen Exchange: Quantifying Arginine Side-Chain Interactions in Solution.

Project description:The rate with which labile backbone hydrogen atoms in proteins exchange with the solvent has long been used to probe protein interactions in aqueous solutions. Arginine, an essential amino acid found in many interaction interfaces, is capable of an impressive range of interactions via its guanidinium group. The hydrogen exchange rate of the guanidinium hydrogens therefore becomes an important measure to quantify side-chain interactions. Herein we present an NMR method to quantify the hydrogen exchange rates of arginine side-chain 1 Hϵ protons and thus present a method to gauge the strength of arginine side-chain interactions. The method employs 13 C-detection and the one-bond deuterium isotope shift observed for 15 Nϵ to generate two exchanging species in 1 H2 O/2 H2 O mixtures. An application to the protein T4 Lysozyme is shown, where protection factors calculated from the obtained exchange rates correlate well with the interactions observed in the crystal structure. The methodology presented provides an important step towards characterising interactions of arginine side-chains in enzymes, in phase separation, and in protein interaction interfaces in general.

| S-EPMC6391956 | biostudies-literature

Universal quenching of common fluorescent probes by water and alcohols.

Project description:Although biological imaging is mostly performed in aqueous media, it is hardly ever considered that water acts as a classic fluorescence quencher for organic fluorophores. By investigating the fluorescence properties of 42 common organic fluorophores recommended for biological labelling, we demonstrate that H2O reduces their fluorescence quantum yield and lifetime by up to threefold and uncover the underlying fluorescence quenching mechanism. We show that the quenching efficiency is significantly larger for red-emitting probes and follows an energy gap law. The fluorescence quenching finds its origin in high-energy vibrations of the solvent (OH groups), as methanol and other linear alcohols are also found to quench the emission, whereas it is restored in deuterated solvents. Our observations are consistent with a mechanism by which the electronic excitation of the fluorophore is resonantly transferred to overtones and combination transitions of high-frequency vibrational stretching modes of the solvent through space and not through hydrogen bonds. Insight into this solvent-assisted quenching mechanism opens the door to the rational design of brighter fluorescent probes by offering a justification for protecting organic fluorophores from the solvent via encapsulation.

| S-EPMC8179231 | 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