Red light, green light: probing single molecules using alternating-laser excitation

2008 ◽  
Vol 36 (4) ◽  
pp. 738-744 ◽  
Author(s):  
Yusdi Santoso ◽  
Ling Chin Hwang ◽  
Ludovic Le Reste ◽  
Achillefs N. Kapanidis
Keyword(s):  
Single Molecule ◽  
Laser Excitation ◽  
Dynamic Range ◽  
Red Light ◽  
Resonance Energy Transfer ◽  
Green Light ◽  
Resonance Energy ◽  
Distance Measurements ◽  
Single Molecule Fret ◽  
Alternating Laser Excitation

Single-molecule fluorescence methods, particularly single-molecule FRET (fluorescence resonance energy transfer), have provided novel insights into the structure, interactions and dynamics of biological systems. ALEX (alternating-laser excitation) spectroscopy is a new method that extends single-molecule FRET by providing simultaneous information about structure and stoichiometry; this new information allows the detection of interactions in the absence of FRET and extends the dynamic range of distance measurements that are accessible through FRET. In the present article, we discuss combinations of ALEX with confocal microscopy for studying in-solution and in-gel molecules; we also discuss combining ALEX with TIRF (total internal reflection fluorescence) for studying surface-immobilized molecules. We also highlight applications of ALEX to the study of protein–nucleic acid interactions.

scholarly journals Cross-validation of distance measurements in proteins by PELDOR/DEER and single-molecule FRET

2020 ◽  
Author(s):  
Martin F. Peter ◽  
Christian Gebhardt ◽  
Rebecca Mächtel ◽  
Janin Glaenzer ◽  
Gavin H. Thomas ◽  
...  
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Protein Interactions ◽  
Large Scale ◽  
Double Resonance ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Resonance Spectroscopy ◽  
Distance Measurements ◽  
Single Molecule Fret

AbstractPulsed electron-electron double resonance spectroscopy (PELDOR or DEER) and single molecule Förster resonance energy transfer spectroscopy (smFRET) are recent additions to the toolbox of integrative structural biology. Both methods are frequently used to visualize conformational changes and to determine nanometer-scale distances in biomacromolecules including proteins and nucleic acids. A prerequisite for the application of PELDOR/DEER and smFRET is the presence of suitable spin centers or fluorophores in the target molecule, which are usually introduced via chemical biology methods. The application portfolio of the two methods is overlapping: each allows determination of distances, to monitor distance changes and to visualize conformational heterogeneity and -dynamics. Both methods can provide qualitative information that facilitates mechanistic understanding, for instance on conformational changes, as well as quantitative data for structural modelling. Despite their broad application, a comprehensive comparison of the accuracy of PELDOR/DEER and smFRET is still missing and we set out here to fill this gap. For this purpose, we prepared a library of double cysteine mutants of three well-studied substrate binding proteins that undergo large-scale conformational changes upon ligand binding. The distances between the introduced spin- or fluorescence labels were determined via PELDOR/DEER and smFRET, using established standard experimental protocols and data analysis routines. The experiments were conducted in the presence and absence of the natural ligands to investigate how well the ligand-induced conformational changes could be detected by the two methods. Overall, we found good agreement for the determined distances, yet some surprising inconsistencies occurred. In our set of experiments, we identified the source of discrepancies as the use of cryoprotectants for PELDOR/DEER and label-protein interactions for smFRET. Our study highlights strength and weaknesses of both methods and paves the way for a higher confidence in quantitative comparison of PELDOR/DEER and smFRET results in the future.

SOLVING SINGLE BIOMOLECULES BY ADVANCED FRET-BASED SINGLE-MOLECULE FLUORESCENCE TECHNIQUES

2013 ◽  
Vol 08 (03n04) ◽  
pp. 161-190 ◽  
Author(s):  
M. J. RUEDAS-RAMA ◽  
J. M. ALVAREZ-PEZ ◽  
A. ORTE
Keyword(s):  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Coincidence Detection ◽  
Special Issue ◽  
Single Molecule Fluorescence ◽  
Single Molecule Fret ◽  
Technical Developments ◽  
Orthogonal Dimension ◽  
Alternating Laser Excitation

The use of Förster resonance energy transfer (FRET) has undergone a renaissance in the last two decades, especially in the study of structure of biomolecules, biomolecular interactions, and dynamics. Thanks to powerful advances in single-molecule fluorescence (SMF) techniques, seeing molecules at work is a reality, which has helped to build up the mindset of molecular machines. In the last few years, many technical developments have broadened the applications of SMF-FRET, expanding the amount of information that can be recovered from individual molecules. Here, we focus on the non-standard SMF-FRET techniques, such as two-color coincidence detection (TCCD), alternating laser excitation (ALEX), multiparameter fluorescence detection (MFD); the addition of fluorescence lifetime as an orthogonal dimension in single-molecule experiments; or the development of novel and improved methods of analysis constituting to a set of advanced methodologies that may become routine tools in a close future. [Formula: see text]Special Issue Comment: This review about advanced single-molecule FRET techniques is specially related to the review by Jørgensen and Hatzakis,6 who detail experimetal strategies to solve the activity of single enzymes. The advanced techniques described in our paper may serve as interesting alternatives when applied to enzyme studies. Our manuscript is also related to the reviews in this Special Issue that deal with model solving.22,130

Single-molecule FRET for Ultrasensitive Detection of Biomolecules

2014 ◽  
Vol 1 (1) ◽  
Author(s):  
Kun Yang ◽  
Yong Yang ◽  
Chun-yang Zhang
Keyword(s):  
Single Molecule ◽  
New Technologies ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Molecular Probes ◽  
Ultrasensitive Detection ◽  
Single Molecule Fret ◽  
Biological Reaction ◽  
Spatio Temporal ◽  
Detection Of Biomolecules

AbstractSingle-molecule Förster resonance energy transfer (sm- FRET) has been widely employed to detect biomarkers and to probe the structure and dynamics of biomolecules. By monitoring the biological reaction in a spatio-temporal manner, smFRET can reveal the transient intermediates of biological processes that cannot be obtained by conventional ensemble measurements. This review provides an overview of singlemolecule FRET and its applications in ultrasensitive detection of biomolecules, including the major techniques and the molecular probes used for smFRET as well as the biomedical applications of smFRET. Especially, the combination of sm- FRET with new technologies might expand its applications in clinical diagnosis and biomedical research

scholarly journals Single-molecule FRET monitors CLC transporter conformation and subunit independence

2020 ◽  
Author(s):  
Ricky C. Cheng ◽  
Ayush Krishnamoorti ◽  
Vladimir Berka ◽  
Ryan J Durham ◽  
Vasanthi Jayaraman ◽  
...  
Keyword(s):  
Conformational Change ◽  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Chloride Ions ◽  
Conformational State ◽  
Triple Mutant ◽  
Transport Pathways ◽  
Single Molecule Fret ◽  
Extracellular Side

Abstract“CLC” transporters catalyze the exchange of chloride ions for protons across cellular membranes. As secondary active transporters, CLCs must alternately allow ion access to and from the extracellular and intracellular sides of the membrane, adopting outward-facing and inward-facing conformational states. Here, we use single-molecule Förster resonance energy transfer (smFRET) to monitor the conformational state of CLC-ec1, an E. coli homolog for which high-resolution structures of occluded and outward-facing states are known. Since each subunit within the CLC homodimer contains its own transport pathways for chloride and protons, we developed a labeling strategy to follow conformational change within a subunit, without crosstalk from the second subunit of the dimer. Using this strategy, we evaluated smFRET efficiencies for labels positioned on the extracellular side of the protein, to monitor the status of the outer permeation pathway. When [H+] is increased to enrich the outward-facing state, the smFRET efficiencies for this pair decrease. In a triple-mutant CLC-ec1 that mimics the protonated state of the protein and is known to favor the outward-facing conformation, the lower smFRET efficiency is observed at both low and high [H+]. These results confirm that the smFRET assay is following the transition to the outward-facing state and demonstrate the feasibility of using smFRET to monitor the relatively small (~1 Å) motions involved in CLC transporter conformational change. Using the smFRET assay, we show that the conformation of the partner subunit does not influence the conformation of the subunit being monitored by smFRET, thus providing evidence for the independence of the two subunits in the transport process.SUMMARYCheng, Krishnamoorti et al. use single-molecule Förster energy resonance transfer measurements to monitor the conformation of a CLC transporter and to show that the conformational state is not influenced by the neighboring subunit.

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.

scholarly journals Camera-based single-molecule FRET detection with improved time resolution

2015 ◽  
Vol 17 (41) ◽  
pp. 27862-27872 ◽  
Author(s):  
Shazia Farooq ◽  
Johannes Hohlbein
Keyword(s):  
Probability Distribution ◽  
Single Molecule ◽  
Time Resolution ◽  
Laser Excitation ◽  
Single Molecule Detection ◽  
Distribution Analysis ◽  
Single Molecule Fret ◽  
Alternating Laser Excitation

Here the authors report on significant improvements in time-resolution and throughput in camera-based single-molecule detection by combining stroboscopic alternating-laser excitation with dynamic probability distribution analysis.

Single molecule fluorescence resonance energy transfer scanning near-field optical microscopy: potentials and challenges

2015 ◽  
Vol 184 ◽  
pp. 51-69 ◽  
Author(s):  
S. K. Sekatskii ◽  
K. Dukenbayev ◽  
M. Mensi ◽  
A. G. Mikhaylov ◽  
E. Rostova ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Single Molecule ◽  
Near Field ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Nitrogen Vacancy ◽  
Single Molecule Fluorescence ◽  
Single Molecule Fret ◽  
Fluorescence Resonance

A few years ago, single molecule Fluorescence Resonance Energy Transfer Scanning Near-Field Optical Microscope (FRET SNOM) images were demonstrated using CdSe semiconductor nanocrystal–dye molecules as donor–acceptor pairs. Corresponding experiments reveal the necessity to exploit much more photostable fluorescent centers for such an imaging technique to become a practically used tool. Here we report the results of our experiments attempting to use nitrogen vacancy (NV) color centers in nanodiamond (ND) crystals, which are claimed to be extremely photostable, for FRET SNOM. All attempts were unsuccessful, and as a plausible explanation we propose the absence (instability) of NV centers lying close enough to the ND border. We also report improvements in SNOM construction that are necessary for single molecule FRET SNOM imaging. In particular, we present the first topographical images of single strand DNA molecules obtained with fiber-based SNOM. The prospects of using rare earth ions in crystals, which are known to be extremely photostable, for single molecule FRET SNOM at room temperature and quantum informatics at liquid helium temperatures, where FRET is a coherent process, are also discussed.

scholarly journals Sub-millisecond conformational transitions of short single-stranded DNA lattices by photon correlation single-molecule FRET

2021 ◽  
Author(s):  
Brett Israels ◽  
Claire S. Albrecht ◽  
Anson Dang ◽  
Megan Barney ◽  
Peter H. von Hippel ◽  
...  
Keyword(s):  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Correlation ◽  
Distribution Functions ◽  
Ssdna Binding ◽  
Single Molecule Fret ◽  
Thermally Driven ◽  
Ssb Proteins ◽  
Minority Species

Thermally-driven conformational fluctuations (or 'breathing') of DNA plays important roles in the function and regulation of the 'macromolecular machinery of genome expression.' Fluctuations in double-stranded (ds) DNA are involved in the transient exposure of pathways to protein binding sites within the DNA framework, leading to the binding of functional and regulatory proteins to single-stranded (ss) DNA templates. These interactions often require that the ssDNA sequences, as well as the proteins involved, assume transient conformations critical for successful binding. Here we use microsecond-resolved single-molecule F&oumlrster Resonance Energy Transfer (smFRET) experiments to investigate the backbone fluctuations of short (ss) oligo- oligo(dT)n templates within DNA constructs that can also serve as models for ss-dsDNA junctions. Such junctions, as well as the attached ssDNA sequences, are involved in the binding of ssDNA binding (ssb) proteins that control and integrate the mechanisms of DNA replication complexes. We have used these data to determine multi-order time-correlation functions (TCFs) and probability distribution functions (PDFs) that characterize the kinetic and thermodynamic behavior of the system. We find that the oligo(dT)n tails of ss-dsDNA constructs inter-convert, on sub-millisecond time-scales, between three macrostates with distinctly different end-to-end distances. These are: (i) a 'compact' macrostate that represents the dominant species at equilibrium; (ii) a 'partially extended' macrostate that exists as a minority species; and (iii) a 'highly extended' macrostate that is present in trace amounts. We propose a model for ssDNA secondary structure that advances our understanding of how spontaneously formed nucleic acid conformations may facilitate the activities of ssDNA associating proteins.

scholarly journals DeepFRET: Rapid and automated single molecule FRET data classification using deep learning

2020 ◽  
Author(s):  
Johannes Thomsen ◽  
Magnus B. Sletfjerding ◽  
Stefano Stella ◽  
Bijoya Paul ◽  
Simon Bo Jensen ◽  
...  
Keyword(s):  
Deep Learning ◽  
Structural Biology ◽  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Ground Truth ◽  
Real Data ◽  
Ground Truth Data ◽  
Single Molecule Fret ◽  
Human Operators

AbstractSingle molecule Förster Resonance energy transfer (smFRET) is a mature and adaptable method for studying the structure of biomolecules and integrating their dynamics into structural biology. The development of high throughput methodologies and the growth of commercial instrumentation have outpaced the development of rapid, standardized, and fully automated methodologies to objectively analyze the wealth of produced data. Here we present DeepFRET, an automated standalone solution based on deep learning, where the only crucial human intervention in transiting from raw microscope images to histogram of biomolecule behavior, is a user-adjustable quality threshold. Integrating all standard features of smFRET analysis, DeepFRET will consequently output common kinetic information metrics for biomolecules. We validated the utility of DeepFRET by performing quantitative analysis on simulated, ground truth, data and real smFRET data. The accuracy of classification by DeepFRET outperformed human operators and current commonly used hard threshold and reached >95% precision accuracy only requiring a fraction of the time (<1% as compared to human operators) on ground truth data. Its flawless and rapid operation on real data demonstrates its wide applicability. This level of classification was achieved without any preprocessing or parameter setting by human operators, demonstrating DeepFRET’s capacity to objectively quantify biomolecular dynamics. The provided a standalone executable based on open source code capitalises on the widespread adaptation of machine learning and may contribute to the effort of benchmarking smFRET for structural biology insights.

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