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.

1. PBS Buffer Preparation and Chamber Treatment
NOTE: Wear a laboratory coat and disposable gloves when performing wet chemical experiments. Use eye protection when aligning the laser.
<ol>
	<li>PBS buffer preparation
	<ol>
		<li>Dissolve 4.5 g of Na2HPO4, 0.44 g of NaH2PO4, and 3.5 g of NaCl in 400 mL of distilled water. Ensure a pH of 7.5 and sterilize the solution by autoclaving on a liquid cycle for 1 h (depending on the autocl...

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 d...

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 border="0" cellpadding="0" cellspacing="0" width="672">
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			<td height="21" style="height:21px;width:204px;">charcoal</td>
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			<td style="width:183px;">Thermo Scientific</td>
			<td style="width:119px;">28320</td>
			<td>10% solution of Polysorbate 20</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">acceptor DNA strand (High FRET)</td>
			<td style="width:183px;">Integrated DNA Technologies</td>
			<td style="width:119px;">178124895</td>
			<td>5&#180;-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>
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			<td height="42" style="height:42px;width:204px;">acceptor DNA strand (Low FRET)</td>
			<td style="width:183px;">Integrated DNA Technologies</td>
			<td style="width:119px;">177956424</td>
			<td>5&#180;-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>
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		<tr height="42">
			<td height="42" style="height:42px;width:204px;">donor DNA strand</td>
			<td style="width:183px;">Integrated DNA Technologies</td>
			<td style="width:119px;">177951437</td>
			<td>5&#180; -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 height="42">
			<td height="42" style="height:42px;width:204px;">DNA strand (No FRET)</td>
			<td style="width:183px;">Integrated DNA Technologies</td>
			<td style="width:119px;"></td>
			<td>5&#180; -d(CGG CCT ATT TCG GAG TTG TAA ACA GAG ATC GCC TTA AAC GTT CGC CTA GAC TAG TCC AAG TAT TGC)</td>
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			<td style="width:119px;">E6331000025</td>
			<td>nexus gradient</td>
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			<td height="42" style="height:42px;width:204px;">Alexa Fluor 488 C5&#160;Maleimide</td>
			<td style="width:183px;">Thermo Scientific</td>
			<td style="width:119px;">A10254</td>
			<td>termed cyan-green fluorophore in the manuscript</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">Alexa Fluor 647 C2&#160;Maleimide</td>
			<td style="width:183px;">Thermo Scientific</td>
			<td style="width:119px;">A20347</td>
			<td>termed far-red fluorophore in the manuscript</td>
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			<td height="21" style="height:21px;width:204px;">Rhodamine 110</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:119px;">83695-250MG</td>
			<td></td>
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			<td height="21" style="height:21px;width:204px;">Rhodamine 101</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:119px;">83694-500MG</td>
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			<td height="21" style="height:21px;width:204px;">LB Broth, Miller</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">BP1426</td>
			<td>For culture of E. coli</td>
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			<td height="21" style="height:21px;width:204px;">Ampicillin</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:119px;">A0166</td>
			<td>Used at 100 ug/ml final concentration in selective LB medium to maintain plasmid selection</td>
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			<td height="21" style="height:21px;width:204px;">Tetracyline&#160;</td>
			<td style="width:183px;">Calbiochem</td>
			<td style="width:119px;">58346</td>
			<td>Used at 12.5 ug/ml final concentration in selective LB medium to maintain gor (flutathione reductase) mutation in Origami B(DE3) strains to facilitate disulfide bond oxidation</td>
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			<td height="21" style="height:21px;width:204px;">Kanamycin</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">BP906-5</td>
			<td>Used at 15 ug/ml final concentration in selective LB medium to maintain trxB (rhioredoxin reductase) mutation in B(DE3) stains to facilitate disulfide bond oxidation</td>
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			<td height="42" style="height:42px;width:204px;">Origami B(DE3) Competent Cells</td>
			<td style="width:183px;">Millipore</td>
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			<td>Competent E. coli cells for expression of protein with disulfide bridges</td>
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			<td height="42" style="height:42px;width:204px;">Isopropyl-&#946;-D-thiogalactopyranoside (IPTG)</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">BP1755</td>
			<td>For induction of E. coli protein expression</td>
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			<td height="21" style="height:21px;width:204px;">HiTrap Chelating HP</td>
			<td style="width:183px;">GE Life Sciences</td>
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			<td>For Large-scale FPLC Purification of His-tagged protein</td>
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			<td style="width:183px;">Sigma-Aldrich</td>
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			<td align="right" style="width:119px;">28406264</td>
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			<td style="width:167px;">ET525/50m 25 mm diameter mount</td>
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			<td height="21" style="height:21px;width:204px;">Red pass filter</td>
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			<td height="21" style="height:21px;width:204px;">UV-Vis spectrophotometer</td>
			<td style="width:183px;">Varian</td>
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			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Fluorolog 3 fluorometer</td>
			<td style="width:183px;">Horiba</td>
			<td style="width:119px;">FL3-22-R3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Fluorohub TCSPC controller</td>
			<td style="width:183px;">Horiba</td>
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			<td>TCSPC electronics for ensemble measurements</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">NanoLed 485L</td>
			<td style="width:183px;">Horiba</td>
			<td style="width:119px;">485L</td>
			<td>Blue diode laser</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:204px;">NanoLed 635L</td>
			<td style="width:183px;">Horiba</td>
			<td style="width:119px;">635L</td>
			<td>Red diode laser</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Olympus IX73 Microscope</td>
			<td style="width:183px;">Olympus</td>
			<td style="width:119px;">IX73P2F</td>
			<td>Microscope frame</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:204px;">PMA 40 Hybrid Detector</td>
			<td style="width:183px;">PicoQuant GmbH</td>
			<td style="width:119px;">932200, PMA 40</td>
			<td>Optimized for green detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">PMA 50 Hybrid Detector</td>
			<td style="width:183px;">PicoQuant GmbH</td>
			<td style="width:119px;">932201, PMA 50</td>
			<td>Optimized for ed shifter sensitivity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">485nm laser</td>
			<td style="width:183px;">PicoQuant GmbH</td>
			<td style="width:119px;">LDH-D-C-485</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">640nm laser</td>
			<td style="width:183px;">PicoQuant GmbH</td>
			<td style="width:119px;">LDH-D-C-640</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">Hydraharp 400 and TTTR acqusition software</td>
			<td style="width:183px;">PicoQuant</td>
			<td align="right" style="width:119px;">930021</td>
			<td>Picosecond event timer and Time Correlated Single Photon Coutning Unit, includes TTTR acqusition software</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">SEPIA II SLM 828 and SEPIA software</td>
			<td style="width:183px;">PicoQuant</td>
			<td align="right" style="width:119px;">910028</td>
			<td>Laser driver for picosecond pulses , includes SEPIA software controller.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">computer</td>
			<td style="width:183px;">Dell</td>
			<td style="width:119px;">optiplex 7010</td>
			<td>cpu: i7-3770 ram:16GB</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">FRET Positioning and Screening (FPS) software</td>
			<td style="width:183px;">Heinrich Heine Unviersity</td>
			<td style="width:119px;"></td>
			<td>It include the Accesibel Volume clacualtor available at http://www.mpc.hhu.de/software/fps.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">MFD suite</td>
			<td style="width:183px;">Heinrich Heine Unviersity</td>
			<td style="width:119px;"></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.

In typical smFRET experiments using an MFD setup (laser lines: 485 nm at 60 &#181;W and 640 nm at 23 &#181;W, section 5.1), the fluorescence sample is diluted to a low-picomolar concentration (10-12 M = 1 pM) and placed in a confocal microscope, where a sub-nanosecond laser pulse excites labeled molecules freely diffusing through an excitation volume. A typical confocal volume is &#60;4 femtoliters (fL). At such low concentrations, only single molecules are detected one at a ti...

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 ...

The overall goal of this protocol is to perform high precision inter-dye distance measurements using multi-parameter fluorescence detection of F

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Biochemistry

10.6K Görüntüleme.  Clemson University. The overall goal of this protocol is to perform high precision inter-dye distance measurements using multi-parameter fluorescence detection of F