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

2011 ◽  
Vol 39 (2) ◽  
pp. 611-616 ◽  
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.

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

2011 ◽  
Vol 392 (1-2) ◽  
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.

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.

Subunit movement in individual H+-ATP synthases during ATP synthesis and hydrolysis revealed by fluorescence resonance energy transfer

2005 ◽  
Vol 33 (4) ◽  
pp. 878-882 ◽  
Author(s):  
M. Börsch ◽  
P. Gräber
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Single Molecule ◽  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Atp Synthesis ◽  
Fluorescence Resonance ◽  
Atp Synthases

F-type H+-ATP synthases synthesize ATP from ADP and phosphate using the energy supplied by a transmembrane electrochemical potential difference of protons. Rotary subunit movements within the enzyme drive catalysis in either an ATP hydrolysis or an ATP synthesis direction respectively. To monitor these subunit movements and associated conformational changes in real time and with subnanometre resolution, a single-molecule FRET (fluorescence resonance energy transfer) approach has been developed using the double-labelled H+-ATP synthase from Escherichia coli. After reconstitution into a liposome, this enzyme was able to catalyse ATP synthesis when the membrane was energized.

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

2016 ◽  
Author(s):  
Antonino Ingargiola ◽  
Eitan Lerner ◽  
SangYoon Chung ◽  
Shimon Weiss ◽  
Xavier Michalet
Keyword(s):  
Open Source ◽  
Single Molecule ◽  
Conformational Changes ◽  
Single Photon ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Widespread Application ◽  
Single Molecule Fret ◽  
Donor And Acceptor ◽  
Burst Analysis

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.

Structural dynamics of P-type ATPase ion pumps

2019 ◽  
Vol 47 (5) ◽  
pp. 1247-1257 ◽  
Author(s):  
Mateusz Dyla ◽  
Sara Basse Hansen ◽  
Poul Nissen ◽  
Magnus Kjaergaard
Keyword(s):  
Structural Dynamics ◽  
Single Molecule ◽  
New Technologies ◽  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved ◽  
Dynamics Simulations ◽  
Types Of Information ◽  
P Type

Abstract P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.

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

eLife ◽  
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.

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.

Identification of Allosteric Fingerprints of Alpha-Synuclein Aggregates in Matrix Metalloprotease-1 and Substrate-Specific Virtual Screening with Single Molecule Insights

2021 ◽  
Author(s):  
Sumaer Kamboj ◽  
Chase Harms ◽  
Derek Wright ◽  
Anthony Nash ◽  
Lokender Kumar ◽  
...  
Keyword(s):  
Virtual Screening ◽  
Single Molecule ◽  
Conformational Changes ◽  
Single Point ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Alpha Synuclein ◽  
Matrix Metalloproteases ◽  
Single Point Mutation ◽  
Mmp Inhibitor

Abstract Alpha-synuclein (aSyn) has implications in pathological protein aggregations in neurodegeneration. Matrix metalloproteases (MMPs) are broad-spectrum proteases and cleave aSyn, leading to aggregation. Previously, we showed that allosteric communications between the two domains of MMP1 on collagen fibril and fibrin depend on substrates, activity, and ligands. Here we report quantification of allostery using single molecule measurements of MMP1 dynamics on aSyn-induced aggregates by calculating Forster Resonance Energy Transfer (FRET) between two dyes attached to the catalytic and hemopexin domains of MMP1. The two domains of MMP1 prefer open conformations that are inhibited by a single point mutation E219Q of MMP1 and tetracycline, an MMP inhibitor. A two-state Poisson process describes the interdomain dynamics, where the two states and kinetic rates of interconversion between them are obtained from histograms and autocorrelations of FRET values. Since a crystal structure of aSyn-bound MMP1 is not available, we performed molecular docking of MMP1 with aSyn using ClusPro. We simulated MMP1 dynamics using different docking poses and matched the experimental and simulated interdomain dynamics to identify an appropriate pose. We used experimentally validated simulations to define conformational changes at the catalytic site and identify allosteric residues in the hemopexin domain having strong correlations with the catalytic motif residues. We defined Shannon entropy to quantify MMP1 dynamics. We performed virtual screening against a site on selected aSyn-MMP1 binding poses and showed that lead molecules differ between free MMP1 and substrate-bound MMP1. Also, identifying aSyn-specific allosteric residues in MMP1 enabled further selection of lead molecules. In other words, virtual screening needs to take substrates into account for substrate-specific control of MMP1 activity. Molecular understanding of interactions between MMP1 and aSyn-induced aggregates may open up the possibility of degrading aggregates by targeting MMPs.

Sign in / Sign up

Export Citation Format

Share Document