This work was supported by National Institutes of Health grant R15GM135904 (awarded to IM) and National Institutes of Health Grant GM110552 (awarded to DBO).

1. Selection of labeling sites
<ol>
	<li>Identify at least three potential labeling sites to triangulate the putative binding site on the existing protein structures. In this case, SecA, SecYEG, and preprotein attached to SecA through genetic fusion were identified2.
	<ol>
		<li>Choose labeling sites within 25-75 &#197; of the putative binding site and in relatively static regions of the protein, the distance will determine the specific FRET dye pair to be used...

Through the use of the FRET mapping methodology, we identified the signal sequence binding site on the SecA protein. Importantly, the presence of a 3-D crystal structure of the complex greatly facilitated our study. The strength of this mapping methodology lies in the ability to use an existing structure to identify locations for labeling. This methodology cannot be used to determine a 3-D structure; however, determination of structural elements56, refinement of an existing structure<sup class="xr...

For proteins, elucidation of dynamics along with 3-dimensional (3-D) structural knowledge leads to an enhanced understanding of structure-function relationships of biomolecular systems. Structural methods, such as X-ray crystallography and cryogenic electron microscopy, capture a static structure and often require the determination of multiple structures to elucidate aspects of biomolecule binding and dynamics1. This article discusses a solution-based method for mapping global structural elements, such as binding sites or binding interactions, that are potentially more transient and less easily captured by static methods. Strong candidate systems for this methodology are ones in which a 3-D structure has been previously determined by X-ray crystallography, NMR spectroscopy, or other structural methods. In this case, we take advantage of the X-ray crystal structure of the SecA-SecYEG complex, a central player in the protein general secretory pathway, to map the location of a signal peptide binding site using F&#246;rster resonance energy transfer (FRET) prior to the transport of the preprotein across the membrane2. Manipulation of the biological system through genetic modifications coupled with our knowledge of the 3-D structure enabled the determination of the conformation of the signal sequence and early mature region immediately prior to insertion into the channel 3.
FRET involves the radiation-less transfer of energy from one molecule (donor) to another (acceptor) in a distance-dependent fashion that is through space4,5. The efficiency of this transfer is monitored through either a decrease in donor or an increase in acceptor fluorescence intensity. The efficiency of energy transfer can be described as
E = R06/(R06 + R6)
in which the R0 value is the distance at which the transfer is 50% efficient6. The technique has previously been described as a molecular ruler and is effective at determining distances in the 2.5-12 nm range, depending on the identity of the donor-acceptor dyes4,7,8,9. The donor fluorescence intensities and lifetimes with or without acceptor allow determination of transfer efficiencies and consequently, distances5,8. Due to the availability of the technology, sensitivity of the method, and ease of use, FRET has also found broad application in such areas as single-molecule fluorescence spectroscopy and confocal microscopy6. The advent of fluorescent proteins such as green fluorescent protein has made the observation of intracellular dynamics and live-cell imaging relatively facile10,11. Many FRET applications such as these are discussed in detail in this virtual issue.
In this study, we particularly focus on the use of FRET measurements to yield distance values to determine structural details. Previously, FRET measurements have been effectively used to determine the conformation of DNA molecules when bound to protein12,13,14, the internal dynamics of proteins, and protein binding interactions15,16,17. The advantages of this method lie in the ability to determine flexible and dynamic structural elements in a solution with relatively low amounts of material. Significantly, this method is particularly effective when used in conjunction with existing structural information and cannot be used as a means of 3-D structure determination. The method provides the best insight and refinement of structure if the work builds on existing structural information often coupled with computational simulation18,19. Here, the use of distances obtained from steady-state and time-resolved FRET measurements is described to map a binding site, the location of which was not known, on an existing crystallographic structure of the SecA-SecYEG complex, major proteins in the general secretory pathway3.
The general secretory pathway, a highly conserved system from prokaryotes to eukaryotes to archaea, mediates the transport of proteins either across or into the membrane to their functional location in the cell. For Gram-negative bacteria, such as E. coli, the organism used in our study, proteins are inserted into or translocated across the inner membrane to the periplasm. The bacterial SecY channel complex (termed the translocon) coordinates with other proteins to translocate the newly synthesized protein, which is directed to its correct location in the cell through a signal sequence typically located at the N-terminus20,21. For proteins bound for the periplasm, the ATPase SecA protein associates with the exit tunnel of the ribosome, and with the preprotein after approximately 100 residues have been translated22. Along with the SecB chaperone protein, it maintains the preprotein in an unfolded state. SecA binds to the SecYEG translocon, and through many cycles of ATP hydrolysis, facilitates protein transport across the membrane23,24.
SecA is a multi-domain protein that exists in cytosolic and membrane-bound forms. A homodimeric protein in the cytosol, SecA consists of a preprotein binding or cross-linking domain25, two nucleotide-binding domains, a helical wing domain, a helical scaffold domain, and the two helix finger (THF)26,27,28,29 (Figure 1). In previous crystallographic studies of the SecA-SecYEG complex, the location of the THF suggested that it was actively involved in protein translocation and subsequent cross-linking experiments with the signal peptide further established the significance of this region in protein translocation30,31. Previous studies, using the FRET mapping methodology, demonstrated that exogenous signal peptides bind to this region of SecA2,32. To fully understand the conformation and location of the signal sequence and early mature region of the preprotein prior to insertion into the SecYEG channel, a protein chimera in which the signal sequence and residues of the early mature region were attached to SecA through a Ser-Gly linker was created (Figure 1). Using this biologically viable construct, it was further demonstrated that the signal sequence and early mature region of the preprotein bind to the THF in a parallel fashion2. Subsequently, the FRET mapping methodology was used to elucidate the conformation and location of the signal sequence and early mature region in the presence of SecYEG as described below3.
Knowledge of the 3-D structure of the SecA-SecYEG complex33,34,35 and the possible location of the binding site allowed us to judiciously place donor-acceptor labels in locations where the intersection of individual FRET distances identifies the binding site location. These FRET mapping measurements revealed that the signal sequence and the early mature region of the preprotein form a hairpin with the tip located at the mouth of the SecYEG channel, demonstrating that the hairpin structure is templated prior to channel insertion.

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			<td>490 nm LED laser</td>
			<td>Horiba</td>
			<td>1684-LED</td>
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			<td>Alexa Fluor 647 C2 Maleimide//DIBO Alkyne</td>
			<td>Life Technologies</td>
			<td>A20347</td>
			<td></td>
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			<td>Agar</td>
			<td>Difco</td>
			<td>DF0812</td>
			<td></td>
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			<td>Alexa Fluor 488 C5 Maleimide/DIBO Alkyne</td>
			<td>Life Technologies</td>
			<td>A10254</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 DIBO Alkyne</td>
			<td>Life Technologies</td>
			<td>S10904</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 DIBO Alkyne</td>
			<td>Life Technologies</td>
			<td>S10906</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra&#173;4 Centrifugal filter (50kDa MWCO)</td>
			<td>Sigma</td>
			<td>UFC805008</td>
			<td></td>
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		<tr>
			<td>Dodecylmaltoside (DDM)</td>
			<td>Anatrace</td>
			<td>D310</td>
			<td></td>
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			<td>E. coli alkaline phosphatase signal peptide SP22</td>
			<td>Biomolecules Midwest</td>
			<td>N/A</td>
			<td>Synthesized custom item</td>
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			<td>extended signal peptide SP41</td>
			<td>Biomolecules Midwest</td>
			<td>N/A</td>
			<td>Synthesized custom item</td>
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		<tr>
			<td>FluorEssence</td>
			<td>Horiba</td>
			<td>version 2.4</td>
			<td>spectral acquisition program for Fluoromax4 spectrofluorometer</td>
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			<td>Fluoromax 4 spectrofluorometer</td>
			<td>Horiba</td>
			<td>N/A</td>
			<td></td>
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		<tr>
			<td>GlobalsWE</td>
			<td>Laboratory for Fluorescence Dynamics, University of California, Irvine</td>
			<td></td>
			<td>spectral analysis program for time-resolved decays</td>
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			<td>H&#173;4&#173;Azido&#173;Phe&#173;OH</td>
			<td>BACHEM</td>
			<td>4020250.0001</td>
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			<td>LB (Miller) Broth</td>
			<td>Fisher Scientific</td>
			<td>BP9723</td>
			<td></td>
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			<td>Ludox HS-40 colloidal silica (40 wt.% suspension in H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>420816</td>
			<td>dilution is needed to make a proper scattering solution</td>
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			<td>PTI Felix GX</td>
			<td>Horiba</td>
			<td>version 4.1.0.4096</td>
			<td>spectral acquisition program for PTI Time Master Instrument</td>
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		<tr>
			<td>PTI Time Master Instrument</td>
			<td>Horiba</td>
			<td>NA</td>
			<td></td>
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			<td>Pymol Molecular Graphics Program</td>
			<td>Schrodinger</td>
			<td>version 2.4</td>
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			<td>Water bath</td>
			<td>Thermo Scientific</td>
			<td>NESLAB RTE 10</td>
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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.

This study focused on determining the location of the preprotein binding site on SecA prior to insertion of the preprotein into the SecYEG channel. To map the binding site, FRET experiments were performed between different regions of the preprotein and three distinct locations on the SecA and SecYEG proteins (Figure 1A-D). From the distances obtained and three-dimensional structures of SecA, SecYEG, and the preprotein, the location of the preprotein binding ...

Watch this Scientific Journal Video about FÃ¶rster Resonance Energy Transfer Mapping: A New Methodology to Elucidate Global Structural Features at JoVE.com

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

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 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 ...

We have developed a FRET mapping methodology that allows the identification and characterization of ligand binding sites, subunit orientation, conformational changes associated with ligand binding, and dynamic motions of proteins. An advantage of this technique is it can be performed in solution and the molecules can move around freely. The distances measured by this technique are appropriate for biological systems.FRET is broadly applicable to many systems. Mapping enhances the monitoring of structural and dynamic changes in any biomolecular system, and is particularly effective if three-dimensional structural information exists. After purifying the protein, the hardest part is labeling with high efficiency, and removing the free dye.For the experiments, it helps to have as little free dye as possible. To begin, choose labeling sites within 25 to 75 angstrom of the punitive binding site, and in relatively static regions of the protein. The distance will determine the specific FRET dye pair to be used.Locate the labeling sites in protein regions that are relatively distinct from each other. So the sites describe the vertices of a triangle, with the punitive binding site located in the center. To measure the R0 values, depending upon the desired cuvette size and volume, prepare two protein samples at the same concentration of total protein.One with the protein labeled with the donor dye only, and one with the protein labeled with the acceptor dye only. Turn on the fluorometer and open the spectral acquisition and analysis program in the FluorEssence software, if using a spectrofluorometer. Click on the red M to connect the computer to the instrument and choose Emission spectra.Enter scan parameters using the collect experiment menu item, such as excitation wavelength, the range for emission scan, temperature, and sample change your position. Click on RTC and optimized instrument settings as described in the manuscript, by monitoring the fluorescence emission at the peak using an excitation wavelength set at the dye's absorption maximum. Place the donor labeled protein sample in the sample holder, and click run to generate an emission scan of the protein labeled with the donor dye only, by exciting the sample at the dye absorption maximum, and scanning over the emission peak.Establish a baseline for the scan by extending the scan at 25 to 50 nanometers past the end of the peak. Measure the quantum yield of the donor only protein by performing absorptions and fluorescence measurements on samples of different concentrations, as described in the text manuscript. Prepare donor only protein, acceptor only protein, and donor-acceptor protein samples, at the same concentration.Obtain the donor only scan to generate fluorescence emission spectra. Excite the solution at the donor dye absorption maximum, and scan over the donor and acceptor emission peaks. Either exchange the sample to the acceptor only protein, or change the sample change your position to the cuvette containing the acceptor only protein.Obtain an emission scan of the protein labeled with the acceptor dye only. Excite the sample at the donor excitation wavelength. Exchange the sample to the donor-acceptor protein sample or change the sample change your position to the cuvette containing the donor-acceptor labeled protein.Obtain an emission scan of the donor-acceptor protein sample using the same settings. For all spectra, correct background fluorescence by subtracting the background counts measured at the end of the scan. Use a 3D graphical viewing program such as PyMOL to map the distances onto the structure, by directly entering the commands from the script into the command window with the appropriate distance information.Generate a shell for each distance measured, and the associated error. Map the position through the intersection of the different shells. The signal peptide binding site was mapped using three different locations on SecA and SecYEG, and four different locations on the signal peptide.FRET experiments between different regions of the pre-protein and three distinct locations on the SecA and SecYEG proteins map the pre-protein binding site and orientation. The punitive binding site of the signal peptide was previously identified as the two-helix finger, and the SecA C-terminal tail suggested an orientation parallel to the two-helix finger. Steady state FRET spectra between SecA37, and PhoA2, and PhoA22, were obtained.Reduction in the donor intensity indicated that greater energy transferred from PhoA22. Relative to the PhoA2 site, which is located further away from SecA37. Time resolved fluorescence decay spectra demonstrated that the donor-acceptor complex has a shorter homogenous decay consistent with energy transfer associated with one distance.FRET distance shells constructed between the SecA PhoA2 residue, and the triangulated sites, demonstrated that each shell intersects with the two-helix finger, the assumed binding site, which is shown in green. The intersected area is smaller than each FRET shell and includes a large contribution from the helical scaffold. FRET distance shells determined between the PhoA2 residue and the labeled sites, describe a smaller area located closer to the tip of the two-helix finger and the mouth of the SecYEG channel.This intersected area is situated at the opposite end of the two-helix finger from PhoA2. Important things to consider include proper selection of the labeling sites to triangulate the area of interest, to measure the quantum yields for each site, and remove all the free dye. This method aligns with structural information obtained with methods like NMR or X-ray crystallography.When the FRET results are put into a structural context it can enhance the existing information.

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Biochemistry

2.3K vistas.  Wesleyan University. Hemos desarrollado una metodología de mapeo FRET que permite la identificación y caracterización de sitios de unión de ligandos, orientación de subunidades, cambios conformacionales asociados con la unión de ligandos y movimientos dinámicos de proteínas. Una ventaja de esta técnica es que se puede realizar en solución y las moléculas pueden moverse libremente. Las distancias medidas por esta técnica son apropiadas para sistemas biológicos.FRET es ampliamente aplicable a mu...