scholarly journals Methods of peptide conformation studies.

2001 ◽  
Vol 48 (4) ◽  
pp. 1091-1099 ◽  
Author(s):  
A Bierzyński
Keyword(s):  
Hydrogen Exchange ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Peptide Conformation ◽  
Short Review ◽  
Experimental Methods ◽  
Conformational Space ◽  
Conformational Studies ◽  
Leading Role

In solution most of the peptides assume multiple flexible conformations. Determination of the dominant conformers and evaluation of their populations is the aim of peptide conformation studies, in which theoretical and experimental methods play complementary roles. Molecular dynamics or Monte Carlo methods are quite effective in searching the conformational space accessible to a peptide but they are not able to estimate, precisely enough, the populations of various conformations. Therefore, they must be supplemented by experimental data. In this paper, a short review of the experimental methods, most widely used in peptide conformational studies, is presented. Among them NMR plays the leading role. Valuable information is also obtained from hydrogen exchange, fluorescence resonance energy transfer, and circular dichroism measurements. The advantages and shortcomings of these methods are discussed.

scholarly journals Structural Determination of Lipid-bound ApoA-I Using Fluorescence Resonance Energy Transfer

2000 ◽  
Vol 275 (47) ◽  
pp. 37048-37054 ◽  
Author(s):  
Hui-hua Li ◽  
Douglas S. Lyles ◽  
Michael J. Thomas ◽  
Wei Pan ◽  
Mary G. Sorci-Thomas
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Structural Determination ◽  
Fluorescence Resonance

scholarly journals Comparative analysis of the coordinated motion of Hsp70s from different organelles observed by single-molecule three-color FRET

2021 ◽  
Vol 118 (33) ◽  
pp. e2025578118
Author(s):  
Lena Voith von Voithenberg ◽  
Anders Barth ◽  
Vanessa Trauschke ◽  
Benjamin Demarco ◽  
Swati Tyagi ◽  
...  
Keyword(s):  
Single Molecule ◽  
Structural Changes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Conformational Space ◽  
Distribution Analysis ◽  
Nucleotide Exchange ◽  
Nucleotide Analogs ◽  
Recognition Mechanism ◽  
Substrate Peptide

Cellular function depends on the correct folding of proteins inside the cell. Heat-shock proteins 70 (Hsp70s), being among the first molecular chaperones binding to nascently translated proteins, aid in protein folding and transport. They undergo large, coordinated intra- and interdomain structural rearrangements mediated by allosteric interactions. Here, we applied a three-color single-molecule Förster resonance energy transfer (FRET) combined with three-color photon distribution analysis to compare the conformational cycle of the Hsp70 chaperones DnaK, Ssc1, and BiP. By capturing three distances simultaneously, we can identify coordinated structural changes during the functional cycle. Besides the known conformations of the Hsp70s with docked domains and open lid and undocked domains with closed lid, we observed additional intermediate conformations and distance broadening, suggesting flexibility of the Hsp70s in adopting the states in a coordinated fashion. Interestingly, the difference of this distance broadening varied between DnaK, Ssc1, and BiP. Study of their conformational cycle in the presence of substrate peptide and nucleotide exchange factors strengthened the observation of additional conformational intermediates, with BiP showing coordinated changes more clearly compared to DnaK and Ssc1. Additionally, DnaK and BiP were found to differ in their selectivity for nucleotide analogs, suggesting variability in the recognition mechanism of their nucleotide-binding domains for the different nucleotides. By using three-color FRET, we overcome the limitations of the usual single-distance approach in single-molecule FRET, allowing us to characterize the conformational space of proteins in higher detail.

scholarly journals The structural heterogeneity of α-synuclein is governed by several distinct subpopulations with interconversion times slower than milliseconds

2020 ◽  
Author(s):  
Jiaxing Chen ◽  
Sofia Zaer ◽  
Paz Drori ◽  
Joanna Zamel ◽  
Khalil Joron ◽  
...  
Keyword(s):  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Intrinsically Disordered Protein ◽  
Structural Heterogeneity ◽  
Conformational Space ◽  
Conformational Ensemble ◽  
Time Resolved ◽  
Intrinsically Disordered ◽  
Transient Nature

AbstractThe intrinsically disordered protein, α-synuclein, implicated in synaptic vesicle homeostasis and neurotransmitter release, is also associated with several neurodegenerative diseases. The different roles of α-synuclein are characterized by distinct structural states (membrane-bound, dimer, tetramer, oligomer, and fibril), which are originated from its various monomeric conformations. The pathological states, determined by the ensemble of α-synuclein monomer conformations and dynamic pathways of interconversion between dominant states, remain elusive due to their transient nature. Here, we use inter-dye distance distributions from bulk time-resolved Förster resonance energy transfer as restraints in discrete molecular dynamics simulations to map the conformational space of the α-synuclein monomer. We further confirm the generated conformational ensemble in orthogonal experiments utilizing far-UV circular dichroism and cross-linking mass spectrometry. Single-molecule protein-induced fluorescence enhancement measurements show that within this conformational ensemble, some of the conformations of α-synuclein are surprisingly stable, exhibiting conformational transitions slower than milliseconds. Our comprehensive analysis of the conformational ensemble reveals essential structural properties and potential conformations that promote its various functions in membrane interaction or oligomer and fibril formation.

scholarly journals Accurate Determination of Human CPR Conformational Equilibrium by smFRET using Dual Orthogonal Non-Canonical Amino Acid Labeling

2018 ◽  
Author(s):  
Robert B. Quast ◽  
Fataneh Fatemi ◽  
Michel Kranendonk ◽  
Emmanuel Margeat ◽  
Gilles Truan
Keyword(s):  
Single Molecule ◽  
Conformational Equilibrium ◽  
Fluorescent Dyes ◽  
Photophysical Properties ◽  
Accurate Determination ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
General Strategy

ABSTRACTConjugation of fluorescent dyes to proteins - a prerequisite for the study of conformational dynamics by single molecule Förster resonance energy transfer (smFRET) - can lead to substantial changes of the dye’s photophysical properties, ultimately biasing the quantitative determination of inter-dye distances. In particular the popular cyanine dyes and their derivatives, which are by far the most used dyes in smFRET experiments, exhibit such behavior. To overcome this, a general strategy to site-specifically equip proteins with FRET pairs by chemo-selective reactions using two distinct non-canonical amino acids simultaneously incorporated through genetic code expansion in Escherichia coli was developed. Applied to human NADPH- cytochrome P450 reductase (CPR), the importance of homogenously labeled samples for accurate determination of FRET efficiencies was demonstrated. Furthermore, the effect of NADP+ on the ionic strength dependent modulation of the conformational equilibrium of CPR was unveiled. Given its generality and accuracy, the presented methodology establishes a new benchmark to decipher complex molecular dynamics on single molecules.

scholarly journals Extreme mechanical diversity of human telomeric DNA revealed by fluorescence-force spectroscopy

2019 ◽  
Vol 116 (17) ◽  
pp. 8350-8359 ◽  
Author(s):  
Jaba Mitra ◽  
Monika A. Makurath ◽  
Thuy T. M. Ngo ◽  
Alice Troitskaia ◽  
Yann R. Chemla ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Force Spectroscopy ◽  
Telomeric Dna ◽  
Telomeric Sequences ◽  
Substitution Mutant

G-quadruplexes (GQs) can adopt diverse structures and are functionally implicated in transcription, replication, translation, and maintenance of telomere. Their conformational diversity under physiological levels of mechanical stress, however, is poorly understood. We used single-molecule fluorescence-force spectroscopy that combines fluorescence resonance energy transfer with optical tweezers to measure human telomeric sequences under tension. Abrupt GQ unfolding with K+in solution occurred at as many as four discrete levels of force. Added to an ultrastable state and a gradually unfolding state, there were six mechanically distinct structures. Extreme mechanical diversity was also observed with Na+, although GQs were mechanically weaker. Our ability to detect small conformational changes at low forces enabled the determination of refolding forces of about 2 pN. Refolding was rapid and stochastically redistributed molecules to mechanically distinct states. A single guanine-to-thymine substitution mutant required much higher ion concentrations to display GQ-like unfolding and refolded via intermediates, contrary to the wild type. Contradicting an earlier proposal, truncation to three hexanucleotide repeats resulted in a single-stranded DNA-like mechanical behavior under all conditions, indicating that at least four repeats are required to form mechanically stable structures.

A paper-supported sandwich immunosensor based on upconversion luminescence resonance energy transfer for the visual and quantitative determination of a cancer biomarker in human serum

The Analyst ◽  
2020 ◽  
Vol 145 (12) ◽  
pp. 4181-4187 ◽  
Author(s):  
Mengyuan He ◽  
Ning Shang ◽  
Lin Shen ◽  
Zhihong Liu
Keyword(s):  
Energy Transfer ◽  
Quantitative Determination ◽  
Human Serum ◽  
Upconversion Luminescence ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cancer Biomarker

A portable and sensitive paper-supported sandwich immunosensor based on UC-FRET was developed for the visual and quantitative determination of CEA.

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