Quantitative structural information from single-molecule FRET

2015 ◽  
Vol 184 ◽  
pp. 117-129 ◽  
Author(s):  
M. Beckers ◽  
F. Drechsler ◽  
T. Eilert ◽  
J. Nagy ◽  
J. Michaelis
Keyword(s):  
Single Molecule ◽  
Structural Information ◽  
Monte Carlo Sampling ◽  
Dye Molecules ◽  
Distance Information ◽  
Single Molecule Fret ◽  
Single Molecule Studies ◽  
Archaeal Transcription ◽  
Short Time ◽  
Fluorescence Energy

Single-molecule studies can be used to study biological processes directly and in real-time. In particular, the fluorescence energy transfer between reporter dye molecules attached to specific sites on macromolecular complexes can be used to infer distance information. When several measurements are combined, the information can be used to determine the position and conformation of certain domains with respect to the complex. However, data analysis schemes that include all experimental uncertainties are highly complex, and the outcome depends on assumptions about the state of the dye molecules. Here, we present a new analysis algorithm using Bayesian parameter estimation based on Markov Chain Monte Carlo sampling and parallel tempering termed Fast-NPS that can analyse large smFRET networks in a relatively short time and yields the position of the dye molecules together with their respective uncertainties. Moreover, we show what effects different assumptions about the dye molecules have on the outcome. We discuss the possibilities and pitfalls in structure determination based on smFRET using experimental data for an archaeal transcription pre-initiation complex, whose architecture has recently been unravelled by smFRET measurements.

Quantitative single molecule FRET efficiencies using TIRF microscopy

2015 ◽  
Vol 184 ◽  
pp. 131-142 ◽  
Author(s):  
Lasse L. Hildebrandt ◽  
Søren Preus ◽  
Victoria Birkedal
Keyword(s):  
Single Molecule ◽  
Structural Information ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Molecular Complexes ◽  
Distance Information ◽  
Single Molecule Level ◽  
Single Molecule Fret ◽  
Wide Range ◽  
Intermolecular Distances

Förster resonance energy transfer (FRET) microscopy at the single molecule level has the potential to yield information on intra and intermolecular distances within the 2–10 nm range of molecules or molecular complexes that undergo frequent conformation changes. A pre-requirement for obtaining accurate distance information is to determine quantitative instrument independent FRET efficiency values. Here, we applied and evaluated a procedure to determine quantitative FRET efficiencies directly from individual fluorescence time traces of surface immobilized DNA molecules without the need for external calibrants. To probe the robustness of the approach over a wide range of FRET efficiencies we used a set of doubly labelled double stranded DNA samples, where the acceptor position was varied systematically. Interestingly, we found that fluorescence contributions arising from direct acceptor excitation following donor excitation are intrinsically taken into account in these conditions as other correction factors can compensate for inaccurate values of these parameters. We give here guidelines, that can be used through tools within the iSMS software (http://www.isms.au.dk), for determining quantitative FRET and assess uncertainties linked with the procedure. Our results provide insights into the experimental parameters governing quantitative FRET determination, which is essential for obtaining accurate structural information from a wide range of biomolecules.

High pressure single-molecule FRET studies of the lysine riboswitch: cationic and osmolytic effects on pressure induced denaturation

2020 ◽  
Vol 22 (28) ◽  
pp. 15853-15866 ◽  
Author(s):  
Hsuan-Lei Sung ◽  
David J. Nesbitt
Keyword(s):  
High Pressure ◽  
Single Molecule ◽  
Pressure Effects ◽  
Protective Mechanisms ◽  
Single Molecule Fret ◽  
Single Molecule Studies

Protective mechanisms of the piezolyte trimethylamine N-oxide counteracting the pressure effects are revealed by single molecule studies at extreme pressures.

scholarly journals Kinetic and thermodynamic framework for P4-P6 RNA reveals tertiary motif modularity and modulation of the folding preferred pathway

2016 ◽  
Vol 113 (34) ◽  
pp. E4956-E4965 ◽  
Author(s):  
Namita Bisaria ◽  
Max Greenfeld ◽  
Charles Limouse ◽  
Dmitri S. Pavlichin ◽  
Hideo Mabuchi ◽  
...  
Keyword(s):  
Single Molecule ◽  
Structural Changes ◽  
Structural Information ◽  
Equilibrium Constants ◽  
Single Point ◽  
Single Point Mutation ◽  
Single Molecule Fret ◽  
Predictive Understanding ◽  
And Function ◽  
And Behavior

The past decade has seen a wealth of 3D structural information about complex structured RNAs and identification of functional intermediates. Nevertheless, developing a complete and predictive understanding of the folding and function of these RNAs in biology will require connection of individual rate and equilibrium constants to structural changes that occur in individual folding steps and further relating these steps to the properties and behavior of isolated, simplified systems. To accomplish these goals we used the considerable structural knowledge of the folded, unfolded, and intermediate states of P4-P6 RNA. We enumerated structural states and possible folding transitions and determined rate and equilibrium constants for the transitions between these states using single-molecule FRET with a series of mutant P4-P6 variants. Comparisons with simplified constructs containing an isolated tertiary contact suggest that a given tertiary interaction has a stereotyped rate for breaking that may help identify structural transitions within complex RNAs and simplify the prediction of folding kinetics and thermodynamics for structured RNAs from their parts. The preferred folding pathway involves initial formation of the proximal tertiary contact. However, this preference was only ∼10 fold and could be reversed by a single point mutation, indicating that a model akin to a protein-folding contact order model will not suffice to describe RNA folding. Instead, our results suggest a strong analogy with a modified RNA diffusion-collision model in which tertiary elements within preformed secondary structures collide, with the success of these collisions dependent on whether the tertiary elements are in their rare binding-competent conformations.

scholarly journals Spotlighting motors and controls of single FoF1-ATP synthase

2013 ◽  
Vol 41 (5) ◽  
pp. 1219-1226 ◽  
Author(s):  
Michael Börsch ◽  
Thomas M. Duncan
Keyword(s):  
Escherichia Coli ◽  
Atp Synthase ◽  
Single Molecule ◽  
Conformational Changes ◽  
Structural Information ◽  
E Coli ◽  
Single Molecule Fret ◽  
Membrane Enzyme ◽  
Subunit Rotation ◽  
Fof1 Atp Synthase

Subunit rotation is the mechanochemical intermediate for the catalytic activity of the membrane enzyme FoF1-ATP synthase. smFRET (single-molecule FRET) studies have provided insights into the step sizes of the F1 and Fo motors, internal transient elastic energy storage and controls of the motors. To develop and interpret smFRET experiments, atomic structural information is required. The recent F1 structure of the Escherichia coli enzyme with the ϵ-subunit in an inhibitory conformation initiated a study for real-time monitoring of the conformational changes of ϵ. The present mini-review summarizes smFRET rotation experiments and previews new smFRET data on the conformational changes of the CTD (C-terminal domain) of ϵ in the E. coli enzyme.

scholarly journals Single-Molecule FRET Detection of Sub-Nanometer Distance Changes in the Range below a 3-Nanometer Scale

Biosensors ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 168
Author(s):  
Heyjin Son ◽  
Woori Mo ◽  
Jaeil Park ◽  
Joong-Wook Lee ◽  
Sanghwa Lee
Keyword(s):  
Single Molecule ◽  
Short Distance ◽  
High Sensitivity ◽  
Nanometer Scale ◽  
Biological Processes ◽  
Dna Duplexes ◽  
Detection Range ◽  
Distance Range ◽  
Single Molecule Fret ◽  
Fluorescence Energy

Single-molecule fluorescence energy transfer (FRET) detection has become a key technique to monitor intra- and intermolecular distance changes in biological processes. As the sensitive detection range of conventional FRET pairs is limited to 3–8 nm, complement probes are necessary for extending this typical working range. Here, we realized a single-molecule FRET assay for a short distance range of below 3 nm by using a Cy2–Cy7 pair having extremely small spectral overlap. Using two DNA duplexes with a small difference in the labeling position, we demonstrated that our assay can observe subtle changes at a short distance range. High sensitivity in the range of 1–3 nm and compatibility with the conventional FRET assay make this approach useful for understanding dynamics at a short distance.

scholarly journals Single molecule FRET reveals pore size and opening mechanism of a mechano-sensitive ion channel

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Yong Wang ◽  
Yanxin Liu ◽  
Hannah A DeBerg ◽  
Takeshi Nomura ◽  
Melinda Tonks Hoffman ◽  
...  
Keyword(s):  
Single Molecule ◽  
Model System ◽  
Channel Structure ◽  
Fluorescence Energy Transfer ◽  
Mechanosensitive Channels ◽  
Dynamics Model ◽  
Single Molecule Fret ◽  
Direct Observations ◽  
Exact Size ◽  
Fluorescence Energy

The mechanosensitive channel of large conductance, which serves as a model system for mechanosensitive channels, has previously been crystallized in the closed form, but not in the open form. Ensemble measurements and electrophysiological sieving experiments show that the open-diameter of the channel pore is >25 Å, but the exact size and whether the conformational change follows a helix-tilt or barrel-stave model are unclear. Here we report measurements of the distance changes on liposome-reconstituted MscL transmembrane α-helices, using a ‘virtual sorting’ single-molecule fluorescence energy transfer. We observed directly that the channel opens via the helix-tilt model and the open pore reaches 2.8 nm in diameter. In addition, based on the measurements, we developed a molecular dynamics model of the channel structure in the open state which confirms our direct observations.

scholarly journals Yeast Chd1p remodels nucleosomes with unique DNA unwrapping and translocation dynamics

2018 ◽  
Author(s):  
Jaewon Kirk ◽  
Ju Yeon Lee ◽  
Yejin Lee ◽  
Chanshin Kang ◽  
Soochul Shin ◽  
...  
Keyword(s):  
Single Molecule ◽  
Atp Hydrolysis ◽  
Chromatin Remodeler ◽  
Base Pairs ◽  
Nucleosome Remodeling ◽  
Chromatin Remodelers ◽  
Histone Octamer ◽  
Single Molecule Fret ◽  
Single Molecule Studies ◽  
Dna Unwrapping

AbstractChromodomain-helicase-DNA-binding protein 1 (CHD1) remodels chromatin by translocating nucleosomes along DNA, but its mechanism remains poorly understood. Here, we employ a single-molecule fluorescence approach to characterize nucleosome remodeling by yeast CHD1 (Chd1p). We show that Chd1p translocates nucleosomes in steps of multiple base pairs per ATP. ATP binding to Chd1p induces a transient unwrapping of the exit-side DNA, and facilitates nucleosome translocation. ATP hydrolysis induces nucleosome translocation, which is followed by the rewrapping upon the release of the hydrolyzed nucleotide. Multiple Chd1ps binding to a single nucleosome sequentially moves a histone octamer with a preference to the center of DNA fragments, suggesting a new mechanism for regularly spaced nucleosome generation by Chd1p. Our results reveal the unique mechanism by which Chd1p remodels nucleosomes.Significance StatementThere are four major ATP-dependent chromatin remodeler families: SWI/SNF, ISWI, CHD, and INO80/SWR1. The remodeling mechanisms of SWI/SNF and ISWI chromatin remodelers have been elucidated through extensive single-molecule studies, but it remains poorly understood how CHD chromatin remodeler operate. We use single-molecule FRET techniques, and show that Yeast CHD1 uses unique mechanisms to remodel a nucleosome.

scholarly journals Oligomerization of the tetramerization domain of p53 probed by two- and three-color single-molecule FRET

2017 ◽  
Vol 114 (33) ◽  
pp. E6812-E6821 ◽  
Author(s):  
Hoi Sung Chung ◽  
Fanjie Meng ◽  
Jae-Yeol Kim ◽  
Kevin McHale ◽  
Irina V. Gopich ◽  
...  
Keyword(s):  
Single Molecule ◽  
Intrinsically Disordered Proteins ◽  
Dissociation Constants ◽  
Structural Information ◽  
Ensemble Methods ◽  
Disordered Proteins ◽  
Intrinsically Disordered ◽  
Single Molecule Fret ◽  
C Terminus ◽  
Tetramerization Domain

We describe a method that combines two- and three-color single-molecule FRET spectroscopy with 2D FRET efficiency–lifetime analysis to probe the oligomerization process of intrinsically disordered proteins. This method is applied to the oligomerization of the tetramerization domain (TD) of the tumor suppressor protein p53. TD exists as a monomer at subnanomolar concentrations and forms a dimer and a tetramer at higher concentrations. Because the dissociation constants of the dimer and tetramer are very close, as we determine in this paper, it is not possible to characterize different oligomeric species by ensemble methods, especially the dimer that cannot be readily separated. However, by using single-molecule FRET spectroscopy that includes measurements of fluorescence lifetime and two- and three-color FRET efficiencies with corrections for submillisecond acceptor blinking, we show that it is possible to obtain structural information for individual oligomers at equilibrium and to determine the dimerization kinetics. From these analyses, we show that the monomer is intrinsically disordered and that the dimer conformation is very similar to that of the tetramer but the C terminus of the dimer is more flexible.

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