Name: single-molecule förster resonance energy transfer methods for real-time investigation of the holliday junction resolution by gen1
Uploaded: 2019-09-18T13:00:00.000+00:00
Duration: 11 min 27 s
Description: Watch this Scientific Journal Video about Single-Molecule FÃ¶rster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1 at JoVE.com

This work was supported by King Abdullah University of Science and Technology through core funding and Competitive Research Award (CRG3) to S. M. H.

The authors declare no competing financial interests.

In this study,&#160;different smFRET techniques were implemented&#160;to determine the kinetics of HJ resolution by GEN130. Similar smFRET approaches were used to follow the double-flap DNA conformational requirement and cleavage by the DNA replication and repair flap endonuclease 142,43,44. Here, critical steps in this protocol are discussed. The silanization reaction should be free from any trace of humidity. The pegylation solution should be applied rapidly to the silanized glass once PEG is dissolved to avoid hydrolysis. In the multi-channel flow cell, any trapped air in the adhesive sheet should be removed to avoid leakage between neighboring channels. The PCA solution should be freshly prepared since it oxidizes over time. The addition of 10 N NaOH should be dropwise, with vortexing in between. The fluorescence background in the coverslip should be minimal before flowing the fluorescently labeled HJ. The imaging in the flow cell should be performed in one direction to avoid imaging bleached areas. In ALEX experiments, the power of the red laser should be reduced to avoid rapid bleaching of the acceptor. In the time-lapse experiments, the cycle time has to be shorter than the fastest event.
smFRET is a sensitive technique that can provide valuable real-time insights in biomolecular reactions. However, this method has several technical challenges, among which is achieving measurable change in FRET during the biochemical reaction. This is necessary to obtain well-separated features in the histograms and distinguishable states in the time-traces. In many cases, smFRET requires careful design of the substrates, selection of the fluorophore pairs and their positions, and amplification of FRET changes in the DNA substrate because of the little structural changes in the substrate45. Another approach for performing FRET is to use labeled proteins46. The observation window in FRET is limited by the stability of the acceptor such as Cy5 or Alexa Fluor 647 which tends to bleach more rapidly than the donor (Cy3 in this case). Therefore, FRET requires a continuous search for stable fluorophores to extend the experiment duration and efforts to develop oxygen scavenging systems to prolong the fluorescence signal and maximize the signal-to-noise ratio47,48.
Among the tips for troubleshooting in smFRET is balancing the several parameters involved in imaging such as the laser power, exposure time, cycle time, and number of cycles to maximize the fluorescence emission, prolong the experiment duration, and achieve appropriate sampling intervals for the enzyme dynamics. Longer observation times and minimal effects from photobleaching are essential to obtain high fidelity dwell time distributions that represent the enzyme dynamics. ALEX generates better histograms since this method is subjected to lower contributions from photobleached particles compared to single-color FRET. However, the temporal resolution in ALEX is lower than that in single-color FRET.
Finally, smFRET&#8217;s emphasis on detecting conformational/structural changes in individual molecules in real-time bridges the gap between high resolution structural techniques (i.e., X-ray crystallography, nuclear magnetic resonance, electron microscopy), which provides atomic resolution structural details under static conditions and bulk methods that yield the ensemble average of a measurable property. In many aspects, smFRET has proven to be a powerful technique for studying biological systems in real-time.

Single-molecule methods based on fluorescence detection provide high signal-to-noise ratios1. FRET is a spectroscopic technique that can measure distances in the range of 1&#8211;10 nm, rendering this technique as a molecular ruler for measuring distances in the nanometer range2,3. The absorption spectrum of the acceptor has a partial spectral overlap with the donor&#8217;s emission spectrum at the shorter wavelength end. FRET is mediated by the radiation-less energy transfer between a donor and acceptor pair, whereas the efficiency of energy transfer is dependent on the distance and orientation of the acceptor4.
Several approaches have been implemented to minimize the background and improve the detection efficiency of the fluorescence signal5,6. One approach is confocal microscopy, in which a pinhole restricts the excitation spot to a size below the diffraction limit7. Another approach is total internal reflection fluorescence (TIRF), which is a wide-field illumination technique in which the light is directed off-axis above a critical angle8. The light is then totally internally reflected at the interface between the glass and aqueous solution, generating an evanescent wave that only illuminates the fluorophores attached to the glass surface and prevents background from the fluorophores in the rest of the solution.
In confocal microscopy, the molecules can be either freely diffusing or surface immobilized. The attained temporal resolution can be within microseconds to several milliseconds9. The confocal detection for a single molecule is performed by single-photon avalanche diode (SPAD) and point-by-point scanning of the region of interest10. In TIRF, a time-series of a few hundreds of molecules immobilized on the surface is recorded by a position-sensitive two-dimensional charge coupled detector (CCD). The CCD amplifies the fluorescence signal either by intensified phosphor screen and microchannel plate or on-chip multiplication of photoelectrons (EMCCD). The temporal resolution is dependent on the readout speed and quantum efficiency of the CCD and usually on the order of few tens of milliseconds6.
HJ is a central intermediate in DNA repair and recombination11,12,13,14. HJ has two continuous and two crossing strands that connect between the continuous strands without intersecting each other. HJ exists in solution as X-stacked conformers, which undergo continuous isomerization by the continuous strands becoming crossing and the crossing strands becoming continuous in the other conformer15. Isomer preference of the HJ is dependent on the core sequence and ionic environment and has been extensively studied by FRET16,17,18,19.
GEN120 is a monomeric protein in solution21 and requires dimerization to cleave the HJ, thus allowing proper separation of the recombined strands22,23. The stacking conformer preference of the HJ influences the outcome of genetic recombination by setting the orientation of the resolution by the HJ resolvases24. Understanding how GEN1 binds the HJ, coordinates the two incisions, and ensures its full resolution have all been under intensive study21,22,23,25,26,27,28,29,30.
In this study, an objective based TIRF set-up is used as described previously31. Two-color alternating excitation (ALEX) is applied to determine the conformational changes upon the interaction of GEN1 with fluorophore labeled HJ. ALEX produces 2D histograms based on two ratiometric parameters FRET efficiency E, which is donor-acceptor distance-dependent, and the stoichiometry parameter S, which measures the donor-acceptor stoichiometry32. ALEX enables the sorting of fluorescent species based on the stoichiometries of the fluorophores including donor-only, acceptor-only, and mixed subpopulations. ALEX can extend the use of FRET to the full range and can detect differences in fluorophore brightness and oligomerization as well as monitor macromolecule-ligand interactions33.
It is found that GEN1 consistently succeeds in resolving the HJ within the lifetime of the GEN1-HJ complex. The time-dependent conformational changes are derived from the time-traces of individual molecules, while the histograms represent the distribution of the underlying populations. Using time-lapse single-color FRET, fast on-rates and slow off-rates for the GEN1 dimer are demonstrated, which increase the affinity of the assembled GEN1 dimer at the first incision product.

<table><tbody><tr><td>(&amp;plusmn;)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox)</td><td>Sigma-Aldrich</td><td>238813</td><td></td></tr><tr><td>0.1 M sodium bicarbonate buffer</td><td>Fisher</td><td>144-55-8</td><td></td></tr><tr><td>10 % Novex Tris-Borate-EDTA gel</td><td>Thermo Fisher Scientific</td><td>EC6275BOX</td><td></td></tr><tr><td>100 X TIRF objective</td><td>Olympus</td><td>NAPO 1.49</td><td></td></tr><tr><td>3,4-dihroxybenzoic acid (PCA)</td><td>Sigma-Aldrich</td><td>P5630</td><td></td></tr><tr><td>3-aminopropyltriethoxysilane (APTES)</td><td>Sigma-Aldrich</td><td>741442</td><td></td></tr><tr><td>6% Novex Tris-Borate-EDTA gel</td><td>Thermo Fisher Scientific</td><td>EC6265BOX</td><td></td></tr><tr><td>Adhesive sheet</td><td>Grace bio-labs</td><td>SA-S-1L</td><td></td></tr><tr><td>Benchtop refrigerated centrifuge</td><td>Eppendorf</td><td>Z605212</td><td></td></tr><tr><td>Biotin-PEG</td><td>Laysan Bio</td><td>Biotin-PEG-SVA 5000</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>New England Biolabs</td><td>B9001S</td><td></td></tr><tr><td>Calcium Chloride Dihydrate</td><td>Sigma-Aldrich</td><td>31307</td><td></td></tr><tr><td>cation exchange column</td><td>GE healthcare</td><td>MonoS (4.6/100)</td><td></td></tr><tr><td>Cell distruptor</td><td>Constant Cell Disruption System</td><td>TS5/40/CE/GA</td><td></td></tr><tr><td>Coomassie Brilliant Blue</td><td>MP Biomedicals</td><td>808274</td><td></td></tr><tr><td>Cy3 emission filter</td><td>Chroma</td><td>HQ600/40M-25</td><td></td></tr><tr><td>Cy5/Alexa Fluor 647 emission filter</td><td>Chroma</td><td>HQ700/40M-25</td><td></td></tr><tr><td>Dichroic for DV2 filter cube</td><td>Photometrics</td><td>630dcxr-18x26</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Thermo Scientific</td><td>R0861</td><td></td></tr><tr><td>Drill</td><td>Dremel</td><td>200-1/21</td><td></td></tr><tr><td>Electronic cutter</td><td>Copam</td><td>CP-2500</td><td></td></tr><tr><td>EMCCD camera</td><td>Hamamatsu</td><td>C9100-13</td><td></td></tr><tr><td>Epoxy glue</td><td>Devcon</td><td>14250</td><td></td></tr><tr><td>FPLC Aktapurifier UPC 10</td><td>GE Healthcare</td><td>28406268</td><td></td></tr><tr><td>GelQuant.NET software</td><td>biochemlabsolutions.com</td><td>Version 1.8.2</td><td></td></tr><tr><td>GEN1 entry vector</td><td>Harvard plasmid repository</td><td>HSCD00399935</td><td></td></tr><tr><td>Glycerol</td><td>Sigma Life Science</td><td>G5516</td><td></td></tr><tr><td>green laser (emission 532 nm)</td><td>Coherent</td><td>Compass 315M-100</td><td></td></tr><tr><td>Heparin column</td><td>GE healthcare</td><td>HiTrap Heparin column</td><td></td></tr><tr><td>HEPES</td><td>BDH</td><td>BDH4162</td><td></td></tr><tr><td>Image splitter</td><td>Photometrics</td><td>Dualview (DV2)</td><td></td></tr><tr><td>Imidazole</td><td>Sigma-Aldrich</td><td>I2399</td><td></td></tr><tr><td>Inverted microscope</td><td>Olympus</td><td>IX81</td><td></td></tr><tr><td>Isopropyl-&amp;szlig;-D-thiogalactoside (IPTG)</td><td>Goldbio.</td><td>12481C100</td><td></td></tr><tr><td>Laser scanner</td><td>GE healthcare</td><td>Typhoon Trio</td><td></td></tr><tr><td>LB Broth</td><td>Fisher Scientific</td><td>BP1426-500</td><td></td></tr><tr><td>Long pass 532nm filter</td><td>Semrock</td><td>LPD02-532RU-25</td><td></td></tr><tr><td>Magnesium Chloride</td><td>Sigma Life Science</td><td>M8266</td><td></td></tr><tr><td>mPEG</td><td>Laysan Bio</td><td>mPEG-SVA 5000</td><td></td></tr><tr><td>Neutravidin</td><td>Pierce</td><td>31000</td><td></td></tr><tr><td>Ni-NTA column</td><td>GE healthcare</td><td>HisTrap FF</td><td></td></tr><tr><td>NuPAGE 10% Bis-Tris gels</td><td>Novex Life technologies</td><td>NP0301BOX</td><td></td></tr><tr><td>NuPAGE 10% Bis-Tris Protein Gels</td><td>Thermo Fisher Scientific</td><td>NP0302PK2</td><td></td></tr><tr><td>Origin software</td><td>OriginLab Corporation</td><td>Version 8.5</td><td></td></tr><tr><td>Phenylmethylsulfonyl fluoride (PMSF)</td><td>Alexis Biochemicals</td><td>270-184-G025</td><td></td></tr><tr><td>Phosphate-buffered saline</td><td>GIBCO</td><td>14190</td><td></td></tr><tr><td>Polyethylene Tubing (I.D. 0.76 mm O.D. 1.22mm)</td><td>Fisher (Becton Dickinson)</td><td>427416</td><td></td></tr><tr><td>Protocatechuate 3,4-dioxygenase (3,4-PCD)</td><td>Sigma-Aldrich</td><td>P8279-25UN</td><td></td></tr><tr><td>Quad-band dichroic</td><td>Chroma Inc</td><td>Z405/488/532/640rpc</td><td></td></tr><tr><td>red laser (emission 640 nm)</td><td>Coherent</td><td>Cube 640 100C</td><td></td></tr><tr><td>Sodium Chloride</td><td>Fisher Chemical</td><td>S271</td><td></td></tr><tr><td>Sorvall RC-6 plus centrifuge</td><td>Thermo Fisher Scientific</td><td>46910</td><td></td></tr><tr><td>Spectrophotometer</td><td>Thermo Fisher Scientific</td><td>Nanodrop 2000</td><td></td></tr><tr><td>Syringe pump</td><td>Harvard Apparatus</td><td>70-3007</td><td></td></tr><tr><td>Teflon tweezers</td><td>Rubis</td><td>K35A</td><td></td></tr><tr><td>Tris Base</td><td>Promega</td><td>H5135</td><td></td></tr><tr><td>Ultracentrifuge</td><td>Beckman Coulter</td><td>Optima L-90K</td><td></td></tr><tr><td>Ultrafiltration membrane</td><td>Millipore</td><td>UFC90300</td><td></td></tr></tbody></table>

single-molecule f&ouml;rster resonance energy transfer methods for real-time investigation of the holliday junction resolution by gen1

Bulk methods measure the ensemble behavior of molecules, in which individual reaction rates of the underlying steps are averaged throughout the population. Single-molecule F&#246;rster resonance energy transfer (smFRET) provides a recording of the conformational changes taking place by individual molecules in real-time. Therefore, smFRET is powerful in measuring structural changes in the enzyme or substrate during binding and catalysis. This work presents a protocol for single-molecule imaging of the interaction of a four-way Holliday junction (HJ) and gap endonuclease I (GEN1), a cytosolic homologous recombination enzyme. Also presented are single-color and two-color alternating excitation (ALEX) smFRET experimental protocols to follow the resolution of the HJ by GEN1 in real-time. The kinetics of GEN1 dimerization are determined at the HJ, which has been suggested to play a key role in the resolution of the HJ and has remained elusive until now. The techniques described here can be widely applied to obtain valuable mechanistic insights of many enzyme-DNA systems.

Watch this Scientific Journal Video about Single-Molecule FÃ¶rster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1 at JoVE.com

Single-Molecule F&#246;rster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

Presented here is a protocol for performing single-molecule F&#246;rster resonance energy transfer to study HJ resolution. Two-color alternating excitation is used for determining the dissociation constants. Single-color time lapse smFRET is then applied in real-time cleavage assays to obtain the dwell time distribution prior to HJ resolution.

Single-Molecule F&ouml;rster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

Bulk methods measure the ensemble behavior of molecules, in which individual reaction rates of the underlying steps are averaged throughout the ...

single-molecule-frster-resonance-energy-transfer-methods-for-real

Nanyang Technological University

Research

JoVE Journal

Genetics

9.4K Views.  King Abdullah University of Science and Technology (KAUST). Dit protocol stelt ons in staat om de interactiedynamiek en de resolutie van de Holliday-verbinding te monitoren door flap endonuclease 1 op het niveau van één molecuul en andere enzymatische systemen. Nou, sommigen zullen gemiddeld in de hele moleculaire bevolking of beveiligt de onderliggende individuele mechanistische stappen en er is. Single molecule methoden bieden deze details door het toezicht op de real-time gedrag van individuele moleculen.Single molecule methoden kunnen eff...

Video: Single-Molecule Förster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

Biochemistry

Real-time Observation of the DNA Strand Exchange Reaction Mediated by Rad51

The DNA strand exchange reaction mediated by Rad51 is a critical step of homologous recombination. In this reaction, Rad51 forms a nucleoprotein filament on single-stranded DNA (ssDNA) and captures double-stranded DNA (dsDNA) non-specifically to interrogate it for a homologous sequence. After encountering homology, Rad51 catalyzes DNA strand exchange to mediate pairing of the ssDNA with the complementary strand of the dsDNA. This reaction is highly regulated by numerous accessary proteins in vivo. Although conventional biochemical assays have been successfully employed to examine the role of such accessory protein in vitro, kinetic analysis of intermediate formation and its progression into a final product has proven challenging due to the unstable and transient nature of the reaction intermediates. To observe these reaction steps directly in solution, fluorescence resonance energy transfer (FRET)-based real-time observation systems of this reaction were established. Kinetic analysis of real-time observations shows that the DNA strand exchange reaction mediated by Rad51 obeys a three-step reaction model involving the formation of a three-strand DNA intermediate, maturation of this intermediate, and the release of ssDNA from the mature intermediate. The Swi5-Sfr1 complex, an accessary protein conserved in eukaryotes, strongly enhances the second and third steps of this reaction. The FRET-based assays presented here enable us to uncover the molecular mechanisms through which recombination accessary proteins stimulate the DNA strand exchange activity of Rad51. The primary goal of this protocol is to enhance the repertoire of techniques available to researchers in the field of homologous recombination, particularly those working with proteins from species other than Schizosaccharomyces pombe, so that the evolutionary conservation of the findings presented herein can be determined.

Fluorescence resonance energy transfer-based real-time observation systems of the DNA strand exchange reaction mediated by Rad51 were developed. Using the protocols presented here, we are able to detect the formation of reaction intermediates and their conversion into products, while also analyzing the enzymatic kinetics of the reaction.

The DNA strand exchange reaction mediated by Rad51 is a critical step of homologous recombination. In this reaction, Rad51 forms a nucleoprotein filament ...

Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically functional inside the cell, nucleic acid-based therapeutics require an efficient intracellular delivery system. One widely adopted approach is to complex DNA with a gene carrier to form nanocomplexes via electrostatic self-assembly, facilitating cellular uptake of DNA while protecting it against degradation. The challenge lies in the rational design of efficient gene carriers, since premature dissociation or overly stable binding would be detrimental to the cellular uptake and therapeutic efficacy. Nanocomplexes synthesized by bulk mixing showed a diverse range of intracellular unpacking and trafficking behavior, which was attributed to the heterogeneity in size and stability of nanocomplexes. Such heterogeneity hinders the accurate assessment of the self-assembly kinetics and adds to the difficulty in correlating their physical properties to transfection efficiencies or bioactivities. We present a novel convergence of nanophotonics (i.e. QD-FRET) and microfluidics to characterize the real-time kinetics of the nanocomplex self-assembly under laminar flow. QD-FRET provides a highly sensitive indication of the onset of molecular interactions and quantitative measure throughout the synthesis process, whereas microfluidics offers a well-controlled microenvironment to spatially analyze the process with high temporal resolution (~milliseconds). For the model system of polymeric nanocomplexes, two distinct stages in the self-assembly process were captured by this analytic platform. The kinetic aspect of the self-assembly process obtained at the microscale would be particularly valuable for microreactor-based reactions which are relevant to many micro- and nano-scale applications. Further, nanocomplexes may be customized through proper design of microfludic devices, and the resulting QD-FRET polymeric DNA nanocomplexes could be readily applied for establishing structure-function relationships. 

We present a novel and powerful integration of nanophotonics (QD-FRET) and microfluidics to investigate the formation of polyelectrolyte polyplexes, which is expected to provide better control and synthesis of uniform and customizable polyplexes for future nucleic acid-based therapeutics.

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically ...

Biology

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for hundreds of individual restriction endonuclease (REase) molecules in one experiment. This assay does not require protein labeling and can be carried out with a single imaging channel. In addition, the results of multiple individual experiments can be pooled to generate well-populated dwell-time distributions. Analysis of the resulting dwell-time distributions can help elucidate the DNA cleavage mechanism by revealing the presence of kinetic steps that cannot be directly observed. Example data collected using this assay with the well-studied REase, EcoRV - a dimeric Type IIP restriction endonuclease that cleaves the palindromic sequence GAT&#8595;ATC (where &#8595; is the cut site) - are in agreement with prior studies. These results suggest that there are at least three steps in the pathway to DNA cleavage that is initiated by introducing magnesium after EcoRV binds DNA in its absence, with an average rate of 0.17 s-1 for each step.

Using quantum-dot-labeled DNA and total internal reflection fluorescence microscopy, we can investigate the reaction mechanism of restriction endonucleases while using unlabeled protein. This single-molecule technique allows for massively multiplexed observation of individual protein-DNA interactions, and data can be pooled to generate well-populated dwell-time distributions.

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for ...

Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes measurements on freely diffusing biomolecules more accessible. This overview includes a step-by-step protocol for using this instrument to make measurements of precise FRET efficiencies in duplex DNA samples, including details of the sample preparation, instrument setup and alignment, data acquisition, and complete analysis routines. The presented approach, which includes how to determine all the correction factors required for accurate FRET-derived distance measurements, builds on a large body of recent collaborative work across the FRET Community, which aims to establish standard protocols and analysis approaches. This protocol, which is easily adaptable to a range of biomolecular systems, adds to the growing efforts in democratising smFRET for the wider scientific community.

This article provides step-by-step instructions for making fully-corrected accurate FRET measurements on individual, freely diffusing biomolecules using the open-source, inexpensive smfBox, from switch on, through alignment and focusing, to data collection and analysis.

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes ...

F&#246;rster Resonance Energy Transfer Mapping: A New Methodology to Elucidate Global Structural Features

F&#246;rster resonance energy transfer (FRET) is an established fluorescence-based method used to successfully measure distances in and between biomolecules in vitro as well as within cells. In FRET, the efficiency of energy transfer, measured by changes in fluorescence intensity or lifetime, relates to the distance between two fluorescent molecules or labels. Determination of dynamics and conformational changes from the distances are just some examples of applications of this method to biological systems. Under certain conditions, this methodology can add to and enhance existing X-ray crystal structures by providing information regarding dynamics, flexibility, and adaptation to binding surfaces. We describe the use of FRET and associated distance determinations to elucidate structural properties, through the identification of a binding site or the orientations of dimer subunits. Through judicious choice of labeling sites, and often employment of multiple labeling strategies, we have successfully applied these mapping methods to determine global structural properties in a protein-DNA complex and the SecA-SecYEG protein translocation system. In the SecA-SecYEG system, we have used FRET mapping methods to identify the preprotein-binding site and determine the local conformation of the bound signal sequence region. This study outlines the steps for performing FRET mapping studies, including identification of appropriate labeling sites, discussion of possible labels including non-native amino acid residues, labeling procedures, how to perform measurements, and interpreting the data.

The study details the methodology of FRET mapping including the selection of labeling sites, choice of dyes, acquisition, and data analysis. This methodology is effective at determining binding sites, conformational changes, and dynamic motions in protein systems and is most useful if performed in conjunction with existing 3-D structural information.

F&#246;rster resonance energy transfer (FRET) is an established fluorescence-based method used to successfully measure distances in and between ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Automated Two-dimensional Spatiotemporal Analysis of Mobile Single-molecule FRET Probes

Single-molecule F&#246;rster resonance energy transfer (smFRET) is a versatile technique reporting on distances in the sub-nanometer to nanometer range. It has been used in a wide range of biophysical and molecular biological experiments, including the measurement of molecular forces, characterization of conformational dynamics of biomolecules, observation of intracellular colocalization of proteins, and determination of receptor-ligand interaction times. In a widefield microscopy configuration, experiments are typically performed using surface-immobilized probes. Here, a method combining single-molecule tracking with alternating excitation (ALEX) smFRET experiments is presented, permitting the acquisition of smFRET time traces of surface-bound, yet mobile probes in plasma membranes or glass-supported lipid bilayers. For the analysis of recorded data, an automated, open-source software collection was developed supporting (i) the localization of fluorescent signals, (ii) single-particle tracking, (iii) determination of FRET-related quantities including correction factors, (iv) stringent verification of smFRET traces, and (v) intuitive presentation of the results. The generated data can conveniently be used as input for further exploration via specialized software, e.g., for the assessment of the diffusional behavior of probes or the investigation of FRET transitions.

This article presents a method for spatiotemporal analysis of mobile, single-molecule F&#246;rster resonance energy transfer (smFRET)-based probes using widefield fluorescence microscopy. The newly developed software toolkit allows the determination of smFRET time traces of moving probes, including the correct FRET efficiency and the molecular positions, as functions of time.

Single-molecule F&#246;rster resonance energy transfer (smFRET) is a versatile technique reporting on distances in the sub-nanometer to nanometer range. ...

Bioengineering

Studying DNA Looping by Single-Molecule FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.

This study presents a detailed experimental procedure to measure looping dynamics of double-stranded DNA using single-molecule Fluorescence Resonance Energy Transfer (FRET). The protocol also describes how to extract the looping probability density called the J factor.

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into ...

Investigating Interactions Between Histone Modifying Enzymes and Transcription Factors in vivo by Fluorescence Resonance Energy Transfer

Epigenetic regulation of gene expression is commonly affected by histone modifying enzymes (HMEs) that generate heterochromatic or euchromatic histone marks for transcriptional repression or activation, respectively. HMEs are recruited to their target chromatin by transcription factors (TFs). Thus, detecting and characterizing direct interactions between HMEs and TFs are critical for understanding their function and specificity better. These studies would be more biologically relevant if performed in vivo within living tissues. Here, a protocol is described for visualizing interactions in plant leaves between a plant histone deubiquitinase and a plant transcription factor using fluorescence resonance energy transfer (FRET), which allows the detection of complexes between protein molecules that are within &#60;10 nm from each other. Two variations of the FRET technique are presented: SE-FRET (sensitized emission) and AB-FRET (acceptor bleaching), in which the energy is transferred non-radiatively from the donor to the acceptor or emitted radiatively by the donor upon photobleaching of the acceptor. Both SE-FRET and AB-FRET approaches can be adapted easily to discover other interactions between other proteins in planta.

Investigating Interactions Between Histone Modifying Enzymes and Transcription Factors in vivo by Fluorescence Resonance Energy Transfer

Fluorescence resonance energy transfer (FRET) is an imaging technique for detecting protein interactions in living cells. Here, a FRET protocol is presented to study the association of histone-modifying enzymes with transcription factors that recruit them to the target promoters for epigenetic regulation of gene expression in plant tissues.

Epigenetic regulation of gene expression is commonly affected by histone modifying enzymes (HMEs) that generate heterochromatic or euchromatic histone ...

Single-Molecule Förster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

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