FRETBursts: An Open Source Toolkit for Analysis of Freely-Diffusing Single-Molecule FRET

AbstractSingle-molecule Förster Resonance Energy Transfer (smFRET) allows probing intermolecular interactions and conformational changes in biomacromolecules, and represents an invaluable tool for studying cellular processes at the molecular scale. smFRET experiments can detect the distance between two fluorescent labels (donor and acceptor) in the 3–10 nm range. In the commonly employed confocal geometry, molecules are free to diffuse in solution. When a molecule traverses the excitation volume, it emits a burst of photons, which can be detected by single-photon avalanche diode (SPAD) detectors. The intensities of donor and acceptor fluorescence can then be related to the distance between the two fluorophores.While recent years have seen a growing number of contributions proposing improvements or new techniques in smFRET data analysis, rarely have those publications been accompanied by so.ware implementation. In particular, despite the widespread application of smFRET, no complete so.ware package for smFRET burst analysis is freely available to date.In this paper, we introduce FRETBursts, an open source software for analysis of freely-diffusing smFRET data. FRETBursts allows executing all the fundamental steps of smFRET bursts analysis using state-of-the-art as well as novel techniques, while providing an open, robust and welldocumented implementation. Therefore, FRETBursts represents an ideal platform for comparison and development of new methods in burst analysis.We employ modern software engineering principles in order to minimize bugs and facilitate long-term maintainability. Furthermore, we place a strong focus on reproducibility by relying on Jupyter notebooks for FRETBursts execution. Notebooks are executable documents capturing all the steps of the analysis (including data files, input parameters, and results) and can be easily shared to replicate complete smFRET analyzes. Notebooks allow beginners to execute complex workflows and advanced users to customize the analysis for their own needs. By bundling analysis description, code and results in a single document, FRETBursts allows to seamless share analysis workflows and results, encourages reproducibility and facilitates collaboration among researchers in the single-molecule community.

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Single-molecule fluorescence resonance energy transfer techniques on rotary ATP synthases

Biological Chemistry ◽

10.1515/bc.2011.001 ◽

2011 ◽

Vol 392 (1-2) ◽

Cited By ~ 13

Author(s):

Michael Börsch

Keyword(s):

Energy Transfer ◽

Fluorescence Resonance Energy Transfer ◽

Single Molecule ◽

Conformational Changes ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Single Molecule Fret ◽

Intensity Changes ◽

Fluorescence Resonance

Abstract Conformational changes of proteins can be monitored in real time by fluorescence resonance energy transfer (FRET). Two different fluorophores have to be attached to those protein domains which move during function. Distance fluctuations between the fluorophores are measured by relative fluorescence intensity changes or fluorescence lifetime changes. The rotary mechanics of the two motors of FoF1-ATP synthase have been studied in vitro by single-molecule FRET. The results are summarized and perspectives for other transport ATPases are discussed.

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The smfBox: an open-source platform for single-molecule FRET

10.1101/861922 ◽

2019 ◽

Author(s):

Benjamin Ambrose ◽

James Baxter ◽

John Cully ◽

Matthew Willmott ◽

Elliot Steele ◽

...

Keyword(s):

Open Source ◽

Single Molecule ◽

Scientific Community ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Cost Effective ◽

Broad Application ◽

Single Molecule Fret ◽

Open Source Hardware ◽

Acquisition Software

AbstractSingle-molecule Förster Resonance Energy Transfer (smFRET) is a powerful technique capable of resolving both relative and absolute distances within and between structurally dynamic biomolecules. High instrument costs, and a lack of open-source hardware and acquisition software have limited smFRET’s broad application by non-specialists. Here, we present the smfBox, a cost-effective confocal smFRET platform, providing detailed build instructions, open-source acquisition software, and full validation, thereby democratising smFRET for the wider scientific community.

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The smfBox is an open-source platform for single-molecule FRET

Nature Communications ◽

10.1038/s41467-020-19468-4 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Benjamin Ambrose ◽

James M. Baxter ◽

John Cully ◽

Matthew Willmott ◽

Elliot M. Steele ◽

...

Keyword(s):

Open Source ◽

Single Molecule ◽

Scientific Community ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Cost Effective ◽

Broad Application ◽

Single Molecule Fret ◽

Open Source Hardware ◽

Acquisition Software

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

10.1101/2020.11.23.394080 ◽

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.

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Single-molecule FRET reveals nucleotide-driven conformational changes in molecular machines and their link to RNA unwinding and DNA supercoiling

Biochemical Society Transactions ◽

10.1042/bst0390611 ◽

2011 ◽

Vol 39 (2) ◽

pp. 611-616 ◽

Cited By ~ 8

Author(s):

Dagmar Klostermeier

Keyword(s):

Single Molecule ◽

Conformational Changes ◽

Atp Hydrolysis ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Molecular Machines ◽

Dna Supercoiling ◽

Single Molecule Fret ◽

Gyrase A ◽

Rna Unwinding

Many complex cellular processes in the cell are catalysed at the expense of ATP hydrolysis. The enzymes involved bind and hydrolyse ATP and couple ATP hydrolysis to the catalysed process via cycles of nucleotide-driven conformational changes. In this review, I illustrate how smFRET (single-molecule fluorescence resonance energy transfer) can define the underlying conformational changes that drive ATP-dependent molecular machines. The first example is a DEAD-box helicase that alternates between two different conformations in its catalytic cycle during RNA unwinding, and the second is DNA gyrase, a topoisomerase that undergoes a set of concerted conformational changes during negative supercoiling of DNA.

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High-throughput smFRET analysis of freely diffusing nucleic acid molecules and associated proteins

10.1101/651869 ◽

2019 ◽

Author(s):

Maya Segal ◽

Antonino Ingargiola ◽

Eitan Lerner ◽

Sang Yoon Chung ◽

Jonathan A. White ◽

...

Keyword(s):

High Throughput ◽

Single Molecule ◽

Conformational Changes ◽

Single Photon ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Kinetic Studies ◽

Ensemble Averaging ◽

Single Spot ◽

Biologically Relevant

AbstractSingle-molecule Förster resonance energy transfer (smFRET) is a powerful technique for nanometer-scale studies of single molecules. Solution-based smFRET, in particular, can be used to study equilibrium intra- and intermolecular conformations, binding/unbinding events and conformational changes under biologically relevant conditions without ensemble averaging. However, single-spot smFRET measurements in solution are slow. Here, we detail a high-throughput smFRET approach that extends the traditional single-spot confocal geometry to a multispot one. The excitation spots are optically conjugated to two custom silicon single photon avalanche diode (SPAD) arrays. Two-color excitation is implemented using a periodic acceptor excitation (PAX), allowing distinguishing between singly- and doubly-labeled molecules. We demonstrate the ability of this setup to rapidly and accurately determine FRET efficiencies and population stoichiometries by pooling the data collected independently from the multiple spots. We also show how the high throughput of this approach can be used to increase the temporal resolution of single-molecule FRET population characterization from minutes to seconds. Combined with microfluidics, this high-throughput approach will enable simple real-time kinetic studies as well as powerful molecular screening applications.

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Applying Corrections in Single-Molecule FRET

10.1101/083287 ◽

2016 ◽

Cited By ~ 3

Author(s):

Antonino Ingargiola

Keyword(s):

Single Molecule ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Quantum Yields ◽

Physical Parameters ◽

Excitation Scheme ◽

Single Molecule Fret ◽

Direct Excitation ◽

Donor And Acceptor ◽

Analytic Expressions

AbstractSingle-molecule Förster resonance energy transfer (smFRET) experiments can detect the distance between a donor and an acceptor fluorophore on the 3-10nm scale. In ratiometric smFRET experiments, the FRET efficiency is estimated from the ratio of acceptor and total signal (donor + acceptor). An excitation scheme involving two alternating lasers (ALEX) is often employed to discriminate between singly– and doubly-labeled populations thanks to a second ratiometric parameter, the stoichiometry S. Accurate FRET and S estimations requires applying three well-known correction factors: donor emission leakage into the acceptor channel, acceptor direct excitation by the donor excitation laser and the “gamma factor” (i.e. correction for the imbalance between donor and acceptor signals due to different fluorophore’s quantum yields and photon detection effciencies).Expressions to directly correct both raw FRET and S values have been reported in [1] in the context of freely-diffusing smFRET. Here we extend Lee et al. work providing several expressions for the direct excitation coeffcient and highlighting a clear interpretation in terms of physical parameters and experimental quantities. Moreover, we derive a more complete set of analytic expressions for correcting FRET and S. We aim to provide a clear and concise reference for different definitions of correction coeffcients and correction formulas valid for any smFRET experiment both in immobilized and freely-diffusing form.

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Single-molecule FRET for Ultrasensitive Detection of Biomolecules

NanoBioImaging ◽

10.2478/nbi-2013-0002 ◽

2014 ◽

Vol 1 (1) ◽

Cited By ~ 5

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

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Transcription initiation at a consensus bacterial promoter proceeds via a 'bind-unwind-load-and-lock' mechanism

eLife ◽

10.7554/elife.70090 ◽

2021 ◽

Vol 10 ◽

Author(s):

Abhishek Mazumder ◽

Richard H Ebright ◽

Achillefs Kapanidis

Keyword(s):

Single Molecule ◽

Conformational Changes ◽

Transcription Initiation ◽

Core Promoter ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Extensive Study ◽

Active Centre ◽

Transient Nature ◽

Promoter Dna

Transcription initiation starts with unwinding of promoter DNA by RNA polymerase (RNAP) to form a catalytically competent RNAP-promoter complex (RPO). Despite extensive study, the mechanism of promoter unwinding has remained unclear, in part due to the transient nature of intermediates on path to RPo. Here, using single-molecule unwinding-induced fluorescence enhancement to monitor promoter unwinding, and single-molecule fluorescence resonance energy transfer to monitor RNAP clamp conformation, we analyze RPo formation at a consensus bacterial core promoter. We find that the RNAP clamp is closed during promoter binding, remains closed during promoter unwinding, and then closes further, locking the unwound DNA in the RNAP active-centre cleft. Our work defines a new, 'bind-unwind-load-and-lock' model for the series of conformational changes occurring during promoter unwinding at a consensus bacterial promoter and provides the tools needed to examine the process in other organisms and at other promoters.

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Single-molecule FRET monitors CLC transporter conformation and subunit independence

10.1101/2020.09.07.286831 ◽

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.

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