VJ and HS acknowledge support from NIH R01 GM094246 to VJ. HS acknowledges start-up funds from the Clemson University Creative Inquiry Program and the Center for Optical Materials Science and Engineering Technologies at Clemson University. This project was also supported by a training fellowship from the Keck Center for Interdisciplinary Bioscience Training of the Gulf Coast Consortia (NIGMS Grant No. 1 T32GM089657-05) and the Schissler Foundation Fellowship for Translational Studies of Common Human Diseases to DD. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

All the authors declare that they have no competing financial interests with the contents of this article.

In this work, the protocol to align, calibrate, and measure interdye distances with high precision using PIE-MFD single-molecule FRET experiments is presented. By carefully calibrating all instrumental parameters, one can increase the accuracy of the measured distances and reach Angstrom accuracy. To do so, various multidimensional histograms are used to analyze and identify populations for further characterization. Using the mean macro time to verify the stability of the measured samples, it is possible to correct for donor and acceptor photobleaching and to select FRET populations based on the stoichiometry parameter. However, the photophysical properties of the acceptor can change depending on the location of the label. Thus, one can use a SPIE distribution to properly correct for the acceptor quantum yield. Proper photophysical characterization is necessary to determine the gamma factor (&#436;), which, together with other correction factors (e.g., &#945; for crosstalk and &#946; for acceptor excitation with the donor laser), can be used to increase the accuracy of the measured interdye distance. This approach was corroborated using two designed dsDNA standard samples, and an accuracy of ~1 &#197; when compared to expected values was determined.
Different dye selections require adapting the microscope optical elements, such as the dichroic and bandpass filters, to accommodate the proper spectral window of the selected dyes. Accordingly, pulsed lasers need to be selected. More importantly, the selection of dyes is crucial because of several possible photophysical artifacts, such as acceptor and donor bleaching, triplet or dye blinking, or dye sticking to protein surfaces. These artifacts could compromise the interpretation of experimental data. MFD is ideal in this scenario because, by inspecting multiple parameters, it is possible to identify the sources of these artifacts, correct for them, or at least be aware of their existence. The dipole orientation parameter, most of the time assumed as&#160;&#954;2 = 2/3, can cause larger deviations of the determined distance if the dye sticks preferentially to the surface of the biomolecules. Anisotropy of the donor sample, acceptor sample, and donor acceptor can help to resolve whether this assumption is valid or not. In this experiment, it has been found that there is a maximum error of ~2.5% on the measured distance, compared to not making the proper correction and obtaining a 10-20% error. The quantum yield of the acceptor can create a larger source of error. Thus, SPIE is important for addressing this important issue.
It is possible to apply a similar strategy to understand the conformational landscape of the ligand-binding domain of the NMDA receptor to understand the mechanism of agonism on the NMDAR. It was found that the LBD in the presence of an antagonist shuns the accessibility of a high-FRET state, postulated to be responsible for opening the channel73. When comparing experimentally derived distances and the expected values based on crystallographic information, an agreement within 3 &#197; was achieved. More importantly, the new low-populated states can be identified with similar precision.
In summary, single-molecule FRET experiments in MFD mode42 allow one to properly account for experimental artifacts and to derive interdye distance in the range of ~30-70 &#197;. If, instead of a single measured distance, a network of distances is derived, it is possible to use these as restraints in structural modeling, particularly for states that are difficult to characterize with more standard methods of structural biology.

A fundamental goal of structural biology studies is to unravel the relationship between the structure and function of biomolecular machines. The first visual impression of biomolecules (e.g., proteins and nucleic acids) occurred in the 1950s through the development X-ray crystallography1,2. X-ray crystallography provides high-resolution, static structural information constrained by the crystal packing. Therefore, the inherent immobility of X-ray structural models shuns the dynamic nature of biomolecules, a factor that impacts most biological functions3,4,5. Nuclear magnetic resonance (NMR)6,7,8 provided an alternative solution to the problem by resolving structural models in aqueous solutions. A great advantage of NMR is its ability to recover the intrinsic dynamic nature of biomolecules and conformational ensembles, which helps to clarify the intrinsic relationships between structure, dynamics, and function3,4,5. Nevertheless, NMR, limited by sample size and large amounts of sample, requires complex labeling strategies for larger systems. Therefore, there is a pressing need to develop alternative methods in structural biology.
Historically, F&#246;rster resonance energy transfer (FRET)9 has not taken an important role in structural biology because of the misconception that FRET provides low-accuracy distance measurements. It is the purpose of this protocol to revisit the ability of FRET to determine distances on the nanometer scale, such that these distances can be used for building structural models of biomolecules. The first experimental verification of the R-6 dependence on the FRET efficiency was done by Stryer in 196710 by measuring polyprolines of various lengths as a &#34;spectroscopic ruler.&#34; A similar experiment was accomplished at the single-molecule level in 200511. Polyproline molecules turned out to be non-ideal, and thus, double-stranded DNA molecules were later used12. This opened the window for precise distance measurements and the idea of using FRET to identify structural properties of biomolecules.
FRET is optimal when the interdye distance range is from ~0.6-1.3 R0, where R0 is the F&#246;rster distance. For typical fluorophores used in single-molecule FRET experiments, R0 is ~50 &#197;. Typically, FRET offers many advantages over other methods in its ability to resolve and differentiate the structures and dynamics in heterogeneous systems: (i) Due to the ultimate sensitivity of fluorescence, single-molecule FRET experiments13,14,15,16 can resolve heterogeneous ensembles by directly counting and simultaneously characterizing the structures of its individual members. (ii) Complex reaction pathways can be directly deciphered in single-molecule FRET studies because no synchronization of an ensemble is needed. (iii) FRET can access a wide range of temporal domains that span over 10 decades in time, covering a wide variety of biologically relevant dynamics. (iv) FRET experiments can be performed in any solution conditions, in vitro as well as in vivo. The combination of FRET with fluorescence microscopy allows for the study of molecular structures and interactions directly in living cells15,16,17,18,19, even with high precision20. (v) FRET can be applied to systems of nearly any size (e.g., polyproline oligomers21,22,23,24, Hsp9025, HIV reverse transcriptase26, and ribosomes27). (vi) Finally, a network of distances that contains all the dimensionality of biomolecules could be used to derive structural models of static or dynamic molecules18,28,29,30,31,32,33,34,35,36,37.
Therefore, single-molecule FRET spectroscopy can be used to derive distances that are precise enough to be used for distance-restrained structural modeling26. This is possible by taking advantage of multiparameter fluorescence detection (MFD)28,38,39,40,41,42, which utilizes eight dimensions of fluorescence information (i.e., excitation spectrum, fluorescence spectrum, anisotropy, fluorescence lifetime, fluorescence quantum yield, macroscopic time, the fluorescence intensities, and the distance between fluorophores) to accurately and precisely provide distance restraints. Additionally, pulsed interleaved excitation (PIE) is combined with MFD (PIE-MFD)42 to monitor direct excitation acceptor fluorescence and to select single-molecule events arising from samples containing a 1:1 donor-to-acceptor stoichiometry. A typical PIE-MFD setup uses two-pulsed interleaved excitation lasers connected to a confocal microscope body, where photon detection is split into four different channels in different spectral windows and polarization characteristics. More details can be found in Figure 1.
It is important to note that FRET must be combined with computational methods to achieve atomistic-like structural models that are consistent with FRET results26,30. It is not the goal of the present protocol to go over the associated methodology to build structural models with FRET-derived distances. However, these approaches have been applied in combination with other techniques (e.g., small-angle X-ray scattering or electron paramagnetic resonance), giving birth to the field of integrative structural biology43,44,45,46. The current goal is to pave the way for FRET as a quantitative tool in structural biology. As an example, this methodology was used to identify three conformational states in the ligand-binding domain (LBD) of the N-methyl-D-aspartate (NMDA) receptor. The ultimate aim is to overcome the aforementioned limitations and to bring FRET amongst the integrative methods used for the structural determination of biomolecules by providing measured distances with high precision.

<table><tbody><tr><td>charcoal</td><td>Merck KGaA</td><td>K42964486 320</td><td></td></tr><tr><td>syringe filter</td><td>Fisherbrand</td><td>09-719C</td><td>size: 0.20 &amp;micro;m</td></tr><tr><td>chambered coverglass</td><td>Fisher Scientific</td><td>155409</td><td>1.5 borosilicate glass, 8 wells</td></tr><tr><td>microscope cover glass</td><td>Fisher Scientific</td><td>063014-9</td><td>size: 24 x 60-1.5</td></tr><tr><td>Nuclease free water</td><td>Fisher Scientific</td><td>148859</td><td>nuclease free</td></tr><tr><td>tween-20</td><td>Thermo Scientific</td><td>28320</td><td>10% solution of Polysorbate 20</td></tr><tr><td>acceptor DNA strand (High FRET)</td><td>Integrated DNA Technologies</td><td>178124895</td><td>5&amp;acute;-d(CGG CCT ATT TCG GAG TTG TAA ACA GAG AT(Cy5)C GCC TTA AAC GTT CGC CTA GAC TAG TCC AAG TAT TGC)</td></tr><tr><td>acceptor DNA strand (Low FRET)</td><td>Integrated DNA Technologies</td><td>177956424</td><td>5&amp;acute;-d(CGG CCT ATT TCG GAG TTG TAA ACA GAG ATC GCC TT(Cy5)A AAC GTT CGC CTA GAC TAG TCC AAG TAT TGC)</td></tr><tr><td>donor DNA strand</td><td>Integrated DNA Technologies</td><td>177951437</td><td>5&amp;acute; -d(GCA ATA CTT GGA CTA GTC TAG GCG AAC GTT TAA GGC GAT CTC TGT TT(Alexa488)A CAA CTC CGA AAT AGG CCG)</td></tr><tr><td>DNA strand (No FRET)</td><td>Integrated DNA Technologies</td><td></td><td>5&amp;acute; -d(CGG CCT ATT TCG GAG TTG TAA ACA GAG ATC GCC TTA AAC GTT CGC CTA GAC TAG TCC AAG TAT TGC)</td></tr><tr><td>thermal cycler</td><td>Eppendorf</td><td>E6331000025</td><td>nexus gradient</td></tr><tr><td>Alexa Fluor 488 C5&amp;nbsp;Maleimide</td><td>Thermo Scientific</td><td>A10254</td><td>termed cyan-green fluorophore in the manuscript</td></tr><tr><td>Alexa Fluor 647 C2&amp;nbsp;Maleimide</td><td>Thermo Scientific</td><td>A20347</td><td>termed far-red fluorophore in the manuscript</td></tr><tr><td>Rhodamine 110</td><td>Sigma-Aldrich</td><td>83695-250MG</td><td></td></tr><tr><td>Rhodamine 101</td><td>Sigma-Aldrich</td><td>83694-500MG</td><td></td></tr><tr><td>LB Broth, Miller</td><td>Fisher Scientific</td><td>BP1426</td><td>For culture of &lt;em&gt;E. coli&lt;/em&gt;</td></tr><tr><td>Ampicillin</td><td>Sigma-Aldrich</td><td>A0166</td><td>Used at 100 &amp;micro;g/mL final concentration in selective LB medium to maintain plasmid selection</td></tr><tr><td>Tetracyline&amp;nbsp;</td><td>Calbiochem</td><td>58346</td><td>Used at 12.5 &amp;micro;g/mL final concentration in selective LB medium to maintain gor (flutathione reductase) mutation in Origami B(DE3) strains to facilitate disulfide bond oxidation</td></tr><tr><td>Kanamycin</td><td>Fisher Scientific</td><td>BP906-5</td><td>Used at 15 &amp;micro;g/mL final concentration in selective LB medium to maintain trxB (rhioredoxin reductase) mutation in B(DE3) stains to facilitate disulfide bond oxidation</td></tr><tr><td>Origami B(DE3) Competent Cells</td><td>Millipore</td><td>70837-3</td><td>Competent &lt;em&gt;E. coli&lt;/em&gt; cells for expression of protein with disulfide bridges</td></tr><tr><td>Isopropyl-&amp;beta;-D-thiogalactopyranoside (IPTG)</td><td>Fisher Scientific</td><td>BP1755</td><td>For induction of &lt;em&gt;E. coli&lt;/em&gt; protein expression</td></tr><tr><td>HiTrap Chelating HP</td><td>GE Life Sciences</td><td>17-0409-01</td><td>For Large-scale FPLC Purification of His-tagged protein</td></tr><tr><td>Imidazole</td><td>Sigma-Aldrich</td><td>56749</td><td></td></tr><tr><td>Ni-NTA Agarose&amp;nbsp;</td><td>Qiagen</td><td>30210</td><td></td></tr><tr><td>PD-10 Desalting Column</td><td>GE Life Sciences</td><td>17-0851-01</td><td></td></tr><tr><td>AktaPurifier</td><td>GE Life Sciences</td><td>28406264</td><td>FPLC Instrument</td></tr><tr><td>Dialysis tubing</td><td>Spectrum labs</td><td>132562</td><td>15 kD MWCO 24 mm Flath width, 10 meters/roll</td></tr><tr><td>Dichroics</td><td>Semrock</td><td>FF500/646-Di01-25x36</td><td>500/646 BrightLight</td></tr><tr><td>50/50 Beam splitter polarizer</td><td>Qioptiq Linos&amp;nbsp;</td><td>G33 5743 000</td><td>10 x 10 film polarizer</td></tr><tr><td>Green pass filer</td><td>Chroma</td><td>ET525/50m</td><td>ET525/50m 25 mm diameter mount</td></tr><tr><td>Red pass filter</td><td>Chroma</td><td>ET720/150m</td><td>ET720/150m 25 mm diameter mount</td></tr><tr><td>Power Meter</td><td>ThorLabd</td><td>PM200</td><td></td></tr><tr><td>UV-Vis spectrophotometer</td><td>Varian</td><td>Cary300Bio</td><td></td></tr><tr><td>Fluorolog 3 fluorometer</td><td>Horiba</td><td>FL3-22-R3</td><td></td></tr><tr><td>Fluorohub TCSPC controller</td><td>Horiba</td><td>Fluorohub-B</td><td>TCSPC electronics for ensemble measurements</td></tr><tr><td>NanoLed 485L</td><td>Horiba</td><td>485L</td><td>Blue diode laser</td></tr><tr><td>NanoLed 635L</td><td>Horiba</td><td>635L</td><td>Red diode laser</td></tr><tr><td>Olympus IX73 Microscope</td><td>Olympus</td><td>IX73P2F</td><td>Microscope frame</td></tr><tr><td>PMA 40 Hybrid Detector</td><td>PicoQuant GmbH</td><td>932200, PMA 40</td><td>Optimized for green detection</td></tr><tr><td>PMA 50 Hybrid Detector</td><td>PicoQuant GmbH</td><td>932201, PMA 50</td><td>Optimized for ed shifter sensitivity</td></tr><tr><td>485 nm laser</td><td>PicoQuant GmbH</td><td>LDH-D-C-485</td><td></td></tr><tr><td>640 nm laser</td><td>PicoQuant GmbH</td><td>LDH-D-C-640</td><td></td></tr><tr><td>Hydraharp 400 and TTTR acqusition software</td><td>PicoQuant</td><td>930021</td><td>Picosecond event timer and Time Correlated Single Photon Coutning Unit, includes TTTR acqusition software</td></tr><tr><td>SEPIA II SLM 828 and SEPIA software</td><td>PicoQuant</td><td>910028</td><td>Laser driver for picosecond pulses , includes SEPIA software controller.</td></tr><tr><td>computer</td><td>Dell</td><td>optiplex 7010</td><td>cpu: i7-3770 ram:16GB</td></tr><tr><td>FRET Positioning and Screening (FPS) software</td><td>Heinrich Heine Unviersity</td><td></td><td>It include the Accesibel Volume clacualtor available at http://www.mpc.hhu.de/software/fps.html</td></tr><tr><td>MFD suite</td><td>Heinrich Heine Unviersity</td><td></td><td>It includes the BIFL software package Paris; Margarita for visualization of the multiparameter hisotrams, and Probability Distribution Analysis software availabel at http://www.mpc.hhu.de/software/software-package.html</td></tr></tbody></table>

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.

Watch this Scientific Journal Video about High Precision FRET at Single-molecule Level for Biomolecule Structure Determination at JoVE.com

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

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 ...

high-precision-fret-at-single-molecule-level-for-biomolecule

Nanyang Technological University

Research

JoVE Journal

Biochemistry

10.7K Views.  Clemson University.  Hier wordt een protocol voor hoogwaardige FRET-experimenten op het molecuulniveau voorgesteld. Daarnaast kan deze methodologie worden gebruikt om drie conformatieve staten in het ligand-bindende domein van de N-methyl-D-aspartaat (NMDA) receptor te identificeren. Het bepalen van precieze afstande is de eerste stap in de richting van het opbouwen van structurele modellen op basis van FRET-experimenten. 

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

Structural Information from Single-molecule FRET Experiments Using the Fast Nano-positioning System

Single-molecule F&#246;rster Resonance Energy Transfer (smFRET) can be used to obtain structural information on biomolecular complexes in real-time. Thereby, multiple smFRET measurements are used to localize an unknown dye position inside a protein complex by means of trilateration. In order to obtain quantitative information, the Nano-Positioning System (NPS) uses probabilistic data analysis to combine structural information from X-ray crystallography with single-molecule fluorescence data to calculate not only the most probable position but the complete three-dimensional probability distribution, termed posterior, which indicates the experimental uncertainty. The concept was generalized for the analysis of smFRET networks containing numerous dye molecules. The latest version of NPS, Fast-NPS, features a new algorithm using Bayesian parameter estimation based on Markov Chain Monte Carlo sampling and parallel tempering that allows for the analysis of large smFRET networks in a comparably short time. Moreover, Fast-NPS allows the calculation of the posterior by choosing one of five different models for each dye, that account for the different spatial and orientational behavior exhibited by the dye molecules due to their local environment.
Here we present a detailed protocol for obtaining smFRET data and applying the Fast-NPS. We provide detailed instructions for the acquisition of the three input parameters of Fast-NPS: the smFRET values, as well as the quantum yield and anisotropy of the dye molecules. Recently, the NPS has been used to elucidate the architecture of an archaeal open promotor complex. This data is used to demonstrate the influence of the five different dye models on the posterior distribution.

We present the setup and experimental procedure to obtain smFRET data from large donor-acceptor networks with a TIRF microscope. The step-by-step analysis of these measurements with the Bayesian inference software Fast-NPS yields high-resolved structural information via the application of adapted dye models.

Single-molecule F&#246;rster Resonance Energy Transfer (smFRET) can be used to obtain structural information on biomolecular complexes in real-time. ...

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. Atomic force microscopy (AFM) can address this problem by enabling stretching and relaxation of single protein molecules, which gives direct evidence of specific unfolding and refolding characteristics. AFM-based single-molecule force-spectroscopy (AFM-SMFS) provides a means to consistently measure high-energy conformations in proteins that are not possible in traditional bulk (biochemical) measurements. Although numerous papers were published to show principles of AFM-SMFS, it is not easy to conduct SMFS experiments due to a lack of an exhaustively complete protocol. In this study, we briefly illustrate the principles of AFM and extensively detail the protocols, procedures, and data analysis as a guideline to achieve good results from SMFS experiments. We demonstrate representative SMFS results of single protein mechanical unfolding measurements and we provide troubleshooting strategies for some commonly encountered problems.

We describe the detailed procedures and strategies to measure the mechanical properties and mechanical unfolding pathways of single protein molecules using an atomic force microscope. We also show representative results as a reference for selection and justification of good single protein molecule recordings.

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. ...

Investigating Single Molecule Adhesion by Atomic Force Spectroscopy

Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. At the same time the AFM tip is passivated to prevent unspecific interactions between the tip and the substrate, which is a prerequisite to study single molecules attached to the AFM tip. Analyses to determine the adhesion force, the adhesion length, and the free energy of these molecules on solid surfaces and bio-interfaces are shortly presented and external references for further reading are provided. Example molecules are the poly(amino acid) polytyrosine, the graft polymer PI-g-PS and the phospholipid POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine). These molecules are desorbed from different surfaces like CH3-SAMs, hydrogen terminated diamond and supported lipid bilayers under various solvent conditions. Finally, the advantages of force spectroscopic single molecule experiments are discussed including means to decide if truly a single molecule has been studied in the experiment.

A protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. Procedures and examples to determine the adhesion force and free energy of these molecules on solid supports and bio-interfaces are provided.

Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single ...

Engineering

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled protein polymerization process is always a challenge. Here, we have developed an enzymatic methodology for constructing polymerized protein step by step in a rationally-controlled sequence. In this method, the C-terminus of a protein monomer is NGL for protein conjugation using OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases) 1) while the N-terminus was a cleavable TEV (tobacco etch virus) cleavage site plus an L (ENLYFQ/GL) for temporary N-terminal protecting. Consequently, OaAEP1 was able to add only one protein monomer at a time, and then the TEV protease cleaved the N-terminus between Q and G to expose the NH2-Gly-Leu. Then the unit is ready for next OaAEP1 ligation. The engineered polyprotein is examined by unfolding individual protein domain using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Therefore, this study provides a useful strategy for polyprotein engineering and immobilization.

Here, we present a protocol to conjugate protein monomer by enzymes forming protein polymer with a controlled sequence and immobilize it on the surface for single-molecule force spectroscopy studies.

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled ...

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, such as nucleic acids and proteins. Single molecule FRET (sm-FRET) measurements on immobilized molecules permit long observations of the system -effectively until both dyes photobleach- resulting in time-traces that report on biomolecular dynamics with a broad range of timescales from milliseconds to minutes. To facilitate the acquisition of large number of traces for statistical analyses, the process must be automated and the sample environment should be tightly controlled over the entire measurement time (~12 hours). This is accomplished using an automated scanning confocal microscope that allows the interrogation of thousands of single molecules overnight, and a microfluidic cell that permits the controlled exchange of buffer, with restricted oxygen content and maintains a constant temperature throughout the entire measuring period. Here we show how to assemble the microfluidic device and how to activate its surface for DNA immobilization. Then we explain how to prepare a buffer to maximize the photostability and lifetime of the fluorophores. Finally, we show the steps involved in preparing the setup for the automated acquisition of time-resolved single molecule FRET traces of DNA molecules.

In this article we describe how we obtain FRET traces from individual DNA molecules immobilized to a surface using an automated scanning confocal microscope.

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, ...

Biology

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 ...

Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One &#945;-Synuclein Monomer at a Time

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural dynamics and its contributions to biology. While the FRET-based ruler reports on inter-dye distances in the 3-10 nm range, other spectroscopic techniques, such as protein-induced fluorescence enhancement (PIFE), report on the proximity between a dye and a protein surface in the shorter 0-3 nm range. Regardless of the method of choice, its use in measuring freely-diffusing biomolecules one at a time retrieves histograms of the experimental parameter yielding separate centrally-distributed sub-populations of biomolecules, where each sub-population represents either a single conformation that stayed unchanged within milliseconds, or multiple conformations that interconvert much faster than milliseconds, and hence an averaged-out sub-population. In single-molecule FRET, where the reported parameter in histograms is the inter-dye FRET efficiency, an intrinsically disordered protein, such as the &#945;-Synuclein monomer in buffer, was previously reported as exhibiting a single averaged-out sub-population of multiple conformations interconverting rapidly. While these past findings depend on the 3-10 nm range of the FRET-based ruler, we sought to put this protein to the test using single-molecule PIFE, where we track the fluorescence lifetime of site-specific sCy3-labeled &#945;-Synuclein proteins one at a time. Interestingly, using this shorter range spectroscopic proximity sensor, sCy3-labeled &#945;-Synuclein exhibits several lifetime sub-populations with distinctly different mean lifetimes that interconvert in 10-100 ms. These results show that while &#945;-Synuclein might be disordered globally, it nonetheless attains stable local structures. In summary, in this work we highlight the advantage of using different spectroscopic proximity sensors that track local or global structural changes one biomolecule at a time.

Time-resolved single-molecule protein-induced fluorescence enhancement is a useful fluorescence spectroscopic proximity sensor sensitive to local structural changes in proteins. Here we show it can be used to uncover stable local conformations in &#945;-Synuclein, which is otherwise known as globularly unstructured and unstable when measured using the longer range FRET ruler.

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural ...

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 ...

Time-Resolved Fluorescence Anisotropy from Single Molecules for Characterizing Local Flexibility in Biomolecules

We describe a protocol for conducting time-resolved fluorescence anisotropy at the single-molecule level using confocal microscopy to investigate the local flexibility and dynamics of the deoxyribonucleic acid (DNA)-binding forkhead (FKH) domain of the FoxP1 transcription factor. FoxP1 dimerizes through a three-dimensional domain-swapping (3D-DS) mechanism, forming a disordered intermediate with or without DNA. Since 3D-DS involves an intrinsically disordered region, understanding its behavior is crucial for elucidating the structural and functional properties of FoxP1. Using a single-cysteine-labeled FoxP1, we conducted single-molecule fluorescence anisotropy (smFA) experiments, applying dynamic anisotropy Photon Distribution Analysis (daPDA) and time-resolved anisotropy Burst Variance Analysis (traBVA) approaches to probe local flexibility and dynamics. This protocol provides a detailed, step-by-step guide for smFA measurements, emphasizing time-resolved analyses, variance, and probability distribution techniques to capture structural dynamics across different timescales. This approach enabled us to relate dynamics and heterogeneity to FoxP1 dimerization and DNA binding, highlighting the complex action mechanism that characterizes this transcription factor.

Here, we present the protocol to study the local flexibility and dynamics of biomolecules using time-resolved fluorescence anisotropy at the single-molecule level in confocal microscopy mode.

We describe a protocol for conducting time-resolved fluorescence anisotropy at the single-molecule level using confocal microscopy to investigate the ...

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. ...

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

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