Name: using three-color single-molecule fret to study the correlation of protein interactions
Uploaded: 2018-01-30T14:00:00
Description: Watch this Scientific Journal Video about Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions at JoVE.com

This work is funded by the German Research Foundation (INST 39/969-1) and the European Research Council through the ERC Grant Agreement n. 681891.

1. Setup and Prerequisites
<ol>
	<li>Perform the multi-color smFRET measurements on a prism-type TIRFM. A description of a two-color setup as a JoVE publication is given in reference12.
	<ol>
		<li>Construct a multi-color TIRFM. A general layout is detailed in 9.</li>
		<li>Use switchable, diode pumped solid state continuous wave lasers, which render the use of mechanical shutters in the excitation paths unnecessary.</li>
		<li>Employ an asymmetric, elongated prism that p...

<ol>
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We present the experimental procedure to obtain three-color smFRET data for a complex protein system and a step-by-step description of the analysis of these measurements. This approach offers the unique possibility to directly assess the correlation between multiple interaction sites or conformational changes.
In order to obtain suitable multi-color single-molecule data on proteins it is important to perform reproducible measurements at a low noise level. This can be achieved by using an effic...

Many proteins fulfill their function in dynamic complexes with other molecules, mediated by conformational changes and transient associations on a broad range of timescales1,2,3. Coupled to an external energy source (e.g., ATP) these dynamic interactions can lead to directionality in a functional cycle and ultimately maintain the non-equilibrium steady-state in a cell, the prerequisite for life.
In order to fully understand these molecular machines, a static description guided by structural studies is not sufficient. In addition, it is essential to have knowledge of the underlying kinetic model and to determine the kinetic rate constants. Several existing methods allow researchers to study the dynamics of binary interactions between two molecules of interest, e.g., surface plasmon resonance, relaxation methods with a spectroscopic readout (e.g., jump or stopped-flow techniques), and nuclear magnetic resonance. However, their applicability is in most cases limited to simple two-state systems (e.g., one bound and one unbound state) due to the averaging inherent to bulk experiments. In cases where more states or intermediates are involved, they yield only a complex mixture of the rate constants. Single-molecule methods such as optical or magnetic tweezers or two-color smFRET, i.e., one donor and one acceptor fluorophore, with a surface-immobilized sample can recover the rate constants for all observed conformational changes. However, when it comes to interactions affecting more than one binding site, these methods remain limited and the information on the possible correlation of the two (or more) interactions will only be accessible via indirect conclusions from a set of experiments.
Multi-color smFRET4,5,6,7,8,9 offers the opportunity to study the interaction between these components directly, at real time and under near-physiological conditions10. This permits one to investigate for example, the conformation-dependent binding of a ligand or another protein8,9,11. The overall approach presented here is to label the protein(s) of interest at specific positions, to attach one protein to the surface of the measurement chamber, and to track the fluorescence intensity over time on a prism-type TIRFM (for details see 9,12). The spatial proximity of the different dyes can then be determined from the energy transfer between them. Labeling strategies may vary from protein to protein (reviewed in 13) and guidelines to avoid artifacts in smFRET measurements exist14.
Since a donor dye may transfer energy to different acceptor dyes in a multi-color smFRET experiment, the relative position of all dyes is not accessible from excitation of one dye alone15,16. But in combination with alternating laser excitation (ALEX17, and reviewed in 18) this method provides all spatio-temporal information at sub-second and sub-nanometer resolution.
In principal, high resolution structural information can be achieved by using the inter-dye distances calculated from the combination of all fluorescence intensities in a multi-color smFRET experiment with ALEX. However, here we focus on state identification and separation as well as the extraction of kinetic models, where multi-color smFRET is indispensable. When &#34;only&#34; structure determination by triangulation is desired, a set of simpler two-color smFRET experiments with high signal-to-noise ratio can be performed12,19.
We use the partial fluorescence (<img alt="Equation 1" src="/files/ftp_upload/56896/56896eq1.jpg" />) as a proxy for the energy transfer between two fluorophores7. The PF is calculated from the fluorescence intensity analogous to the FRET efficiency of a two-color experiment:
<img alt="Equation 2" src="/files/ftp_upload/56896/56896eq2.jpg" />
Where, <img alt="Equation 3" src="/files/ftp_upload/56896/56896eq3v2.jpg" /> is the intensity in emission channel em after excitation with color ex, and c is the acceptor with the longest wavelength. Detection channels represent the same position in the sample chamber but record different spectral ranges of the fluorescence light. The same identifier for excitation and emission are used in this protocol (i.e., &#34;blue,&#34;&#160;&#34;green,&#34;&#160;and &#34;red&#34;).
Because of experimental shortcomings the measured fluorescence intensities depend not only on the energy transfer but also on fluorophore and setup properties. In order to obtain the true energy transfer efficiency between two fluorophores, the measured intensities have to be corrected. The following procedure is based on reference9. Correction factors for apparent leakage (lk, i.e., the detection of photons from a fluorophore in a channel designated for another dye) and apparent gamma (ag, i.e., the fluorescence quantum yield of the dye and the detection efficiency of the channel) are obtained from single-molecule traces that show an acceptor bleaching event.
The leakage of the donor dye into every possible acceptor channel is calculated from all data points in the recorded fluorescence traces where the acceptor dye bleached but the donor is still fluorescent (<img alt="Equation 4" src="/files/ftp_upload/56896/56896eq4.jpg" />):
<img alt="Equation 5" src="/files/ftp_upload/56896/56896eq5.jpg" />
The median of the leakage histogram is used as the apparent leakage factor. After correction for leakage, the apparent gamma factor is determined from the same set of traces. It is calculated by dividing the change of fluorescence in the acceptor channel by the change of fluorescence in the donor channel upon bleaching of the acceptor dye:
<img alt="Equation 6" src="/files/ftp_upload/56896/56896eq6.jpg" />
Where c again is the detection channel for the acceptor with the longest wavelength. The median of the resulting distribution is used as the apparent correction factor.
The corrected intensities in each channel are obtained by:
<img alt="Equation 7" src="/files/ftp_upload/56896/56896eq7.jpg" />
The PF is then calculated according to:
<img alt="Equation 8" src="/files/ftp_upload/56896/56896eq8.jpg" />
Different populations can be separated in the multi-dimensional space spanned by the PFs. The position and width of each state is determined by fitting the data with multi-dimensional Gaussian functions. Subsequent optimization of one global HMM based on all PF traces provides a quantitative description of the observed kinetics. Even small changes of the rates are detectable.
HMMs provide a way of inferring a state model from a collection of noisy time traces. The system is considered to be in one of a set of discrete, hidden states at any given time and the actual observation (i.e., the emission) is a probabilistic function of this hidden state20. In the case of TIRFM smFRET data, the emission probabilities bi per state i can be modeled by continuous Gaussian probability density functions. At regularly spaced discrete time points, transitions from one to another state can occur according to the transition probability that is time-invariant and only depends on the current state. The transition matrix A contains these transition probabilities aij between all hidden states. The initial state distribution <img alt="Equation 9" src="/files/ftp_upload/56896/56896eq9.jpg" /> gives the state-specific probabilities <img alt="Equation 10" src="/files/ftp_upload/56896/56896eq10.jpg" /> for the first time point of a time trace. Using a maximum-likelihood approach, these parameters can be optimized to best describe the data with the Forward-Backward and Baum-Welch algorithms20,21. This yields the maximum likelihood estimators (MLE). Finally, the state sequence that most likely produced the trajectory of observations can be inferred with the Viterbi algorithm. In contrast to other HMM analyses of smFRET data24,25,26 we do not use the HMM as a mere &#34;smoothing&#34; of the data but extract the kinetic state model from the data set without the need for fitting dwell time histograms27. HMM analysis is done with in-house scripts using Igor Pro. Implementation of the code is based on reference21. We provide a software kit and exemplary data on our webpage in order to follow sections 5 and 6 of this protocol (https://www.singlemolecule.uni-freiburg.de/software/3d-fret). Full software is available upon request.
Time points in the data with PF &#60;-1 or PF &#62;2 in any detection channel are assigned the minimal emission probability for all states (10-200). This prevents artificial transitions at these data points.
The parameters for the emission probabilities are obtained from the fit of the 3D PF histogram with Gaussian functions as described in step 5.7. These parameters are kept fixed during the optimization of the HMM.
In the presented approach, the initial state distribution vector and the transition matrix are used globally to describe the entire ensemble of traces. They are updated based on all N molecules from the data set according to reference27.
Start parameters for the initial state distribution are determined from 2D projections of the PF histogram (step 5.3) and the transition probabilities are set to 0.05 with the exception of the probabilities to stay in the same state, which are chosen such that the probability to leave a certain state is normalized to unity.
A likelihood profiling method is used to give confidence intervals (CIs) for all transition rates21,22, which serve as meaningful estimates for their uncertainty. To calculate the bounds of the CI for a specific rate, the transition probability of interest is fixed to a value other than the MLE. This yields the test model &#955;&#39;. A likelihood ratio (LR) test of the likelihood <img alt="Equation 16" src="/files/ftp_upload/56896/56896eq16.jpg" /> given the data set 0 is performed according to:
<img alt="Equation 11" src="/files/ftp_upload/56896/56896eq11.jpg" />
The 95% confidence bound for the parameter is reached when LR exceeds 3.841, the 95% quantile of a x2-distribution with one degree of freedom22,23.
The power of the method is demonstrated using the Hsp90. This abundant protein is found in bacteria and eukaryotes and is part of the cellular stress response28. It is a promising drug target in cancer treatment29. Hsp90 is a homodimer with one nucleotide binding pocket in the N-terminal domain of each subunit30. It can undergo transitions between at least two globally distinct conformations, one closed and one N-terminal open, V-shaped conformation19,31,32. The dimeric nature directly raises the question of the interplay between the two nucleotide binding sites in Hsp90.
In the following, we provide a step-by-step protocol for the data acquisition and analysis of a three-color smFRET experiment on yeast Hsp90 and nucleotide. The conformation-dependent binding of fluorescently labeled AMP-PNP (AMP-PNP*, a non-hydrolyzable analog of ATP) is analyzed. The application of the described procedure permits the study of the nucleotide binding and at the same time the conformational changes of Hsp90 and thereby reveals the cooperativity between the two nucleotide binding pockets of Hsp90.

<table border="0" cellpadding="0" cellspacing="0" width="1301">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Setup</td>
			<td style="width:445px;"></td>
			<td style="width:167px;"></td>
			<td style="width:347px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">vibration-damped optical table&#160;&#160;</td>
			<td style="width:445px;">Newport, Irvine, CA, USA</td>
			<td style="width:167px;">RS2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">OBIS 473nm LX 75mW LASER</td>
			<td style="width:445px;">Coherent Inc, Santa Clara, CA, USA</td>
			<td style="width:167px;">1185052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">OBIS 532nm LS 50mW LASER</td>
			<td style="width:445px;">Coherent Inc, Santa Clara, CA, USA</td>
			<td style="width:167px;">1261779</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">OBIS 594nm LS 60mW LASER</td>
			<td style="width:445px;">Coherent Inc, Santa Clara, CA, USA</td>
			<td style="width:167px;">1233470</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">OBIS 637nm LX 140mW LASER</td>
			<td style="width:445px;">Coherent Inc, Santa Clara, CA, USA</td>
			<td style="width:167px;">1196625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">laser control unit</td>
			<td style="width:445px;">Coherent Inc, Santa Clara, CA, USA</td>
			<td style="width:167px;">1234465</td>
			<td style="width:347px;">Scientific Remote</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">aspheric telescope lenses</td>
			<td style="width:445px;">Thorlabs Inc, Newton, New Jersey, USA</td>
			<td style="width:167px;"></td>
			<td>d=25.4mm, f=50mm and f=100mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">CF ex1</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">ZET 473/10</td>
			<td>cleanup filter excitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">CF ex2</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">ZET 532/10</td>
			<td>cleanup filter excitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">CF ex3</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">ZET 594/10</td>
			<td>cleanup filter excitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">CF ex4</td>
			<td style="width:445px;">Thorlabs Inc, Newton, New Jersey, USA</td>
			<td>FL635-10</td>
			<td>cleanup filter excitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DM ex1</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">ZQ594RDC</td>
			<td>dichroic mirror excitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DM ex2</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">570DCXR</td>
			<td>dichroic mirror excitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DM ex3</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">ZQ491RDC</td>
			<td>dichroic mirror excitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">AOTFnC-Vis</td>
			<td style="width:445px;">AA Opto-Electronic, Orsay, France</td>
			<td style="width:167px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">&#955;/4 plate</td>
			<td style="width:445px;">Thorlabs Inc, Newton, New Jersey, USA</td>
			<td style="width:167px;">AQWP05M-600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">CFI Apo TIRF 100x</td>
			<td style="width:445px;">Nikon Instruments Inc, Melville, NY, USA</td>
			<td style="width:167px;"></td>
			<td>high-NA objective</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">piezo focus positioner MIPOS 250 CAP</td>
			<td style="width:445px;">piezosystem jena GmbH, Jena, Germany</td>
			<td style="width:167px;"></td>
			<td>Piezo Controller NV 40/1 CLE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">piezo stepper</td>
			<td style="width:445px;">Newport, Irvine, CA, USA</td>
			<td>PZA12</td>
			<td>PZC200-KT NanoPZ Actuator Kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">achromatic aspheric lenses</td>
			<td style="width:445px;">Qioptiq Photonics GmbH &#38; Co. KG, G&#246;ttingen, Germany</td>
			<td style="width:167px;">G322-304-000</td>
			<td>d=50mm, f=200mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">adjustable optical slit</td>
			<td style="width:445px;">Owis GmbH, Staufen i. Br., Germany</td>
			<td>27.160.1212</td>
			<td>max. aperture 12 x 12 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DM det1</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">T 600 LPXR</td>
			<td>dichroic mirror detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DM det2</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">H 560 LPXR superflat</td>
			<td>dichroic mirror detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DM det3</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">HC BS R635</td>
			<td>dichroic mirror detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">BP det1</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">525/40 BrightLine HC</td>
			<td>bandpass filter detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">BP det2</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">586/20 BrightLine HC</td>
			<td>bandpass filter detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">BP det3</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">631/36 BrightLine HC</td>
			<td>bandpass filter detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">BP det4</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">700/75 ET Bandpass</td>
			<td>bandpass filter detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">optical shutters detection</td>
			<td style="width:445px;">Vincent Associates, Rochester, NY, USA</td>
			<td style="width:167px;">Uniblitz VS25S2T0&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">EMCCD iXon Ultra 897</td>
			<td style="width:445px;">Andor Technology Ltd, Belfast, Northern Ireland</td>
			<td style="width:167px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">digital I/O card, PCIe-6535</td>
			<td style="width:445px;">National Instruments, Austin, Texas, USA</td>
			<td style="width:167px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">syringe pump</td>
			<td style="width:445px;">Harvard Apparatus, Holliston, MA, USA</td>
			<td style="width:167px;">PHD22/2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Name</td>
			<td style="width:445px;">Company</td>
			<td style="width:167px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Flow chamber</td>
			<td style="width:445px;"></td>
			<td style="width:167px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">quartz slides</td>
			<td style="width:445px;">G. Finkenbeiner Inc, Waltham, MA, USA</td>
			<td style="width:167px;"></td>
			<td>Spectrosil2000, h=3mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">TEGADERM film</td>
			<td style="width:445px;">3M Deutschland GmbH, Neuss, Germany</td>
			<td style="width:167px;">1626W</td>
			<td>10 x 12cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">spray adhesive</td>
			<td style="width:445px;">3M Deutschland GmbH, Neuss, Germany</td>
			<td style="width:167px;">Photo Mount 050777</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">glycerol</td>
			<td style="width:445px;">Carl Zeiss AG, Oberkochen, Germany</td>
			<td style="width:167px;">Immersol G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">immersion oil</td>
			<td style="width:445px;">OLYMPUS EUROPA SE &#38; CO. KG, Hamburg, Germany</td>
			<td>IMMOIL-F30CC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">prism</td>
			<td style="width:445px;">Vogelsberger Quarzglastechnik GmbH, Hauzenberg, Germany</td>
			<td style="width:167px;"></td>
			<td>Suprasil1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">aluminium prism holder</td>
			<td style="width:445px;"></td>
			<td style="width:167px;"></td>
			<td>custom built</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">hollow setscrews</td>
			<td style="width:445px;">Thorlabs Inc, Newton, New Jersey, USA</td>
			<td style="width:167px;"></td>
			<td>with custom drilling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Tygon S3 E-3603 tubing</td>
			<td style="width:445px;">neoLab Migge GmbH, Heidelberg, Germany</td>
			<td style="width:167px;">2-4450</td>
			<td>ACF00001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">PTFE tubing</td>
			<td style="width:445px;">Bohlender GmbH, Gr&#252;nsfeld, Germany</td>
			<td style="width:167px;">S1810-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Name</td>
			<td style="width:445px;">Company</td>
			<td style="width:167px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Sample</td>
			<td style="width:445px;"></td>
			<td style="width:167px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">yeast Hsp90 D61C, Q385C_biotin</td>
			<td style="width:445px;"></td>
			<td style="width:167px;"></td>
			<td>UniProt ID P02829</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Maleimide derivatives of Atto488, Atto550</td>
			<td style="width:445px;">ATTO-TEC GmbH, Siegen, Germany</td>
			<td style="width:167px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">AMP-PNP*</td>
			<td style="width:445px;">Jena Bioscience, Jena, Germany</td>
			<td style="width:167px;"></td>
			<td style="width:347px;">&#947;-[(6-Aminohexyl)-imido]-AMP-PNP-Atto647N</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Fluospheres</td>
			<td style="width:445px;">Thermo Fisher Scientific, Waltham, MA, USA</td>
			<td style="width:167px;">F8764</td>
			<td>amine-modified, 0.2 &#956;m, yellow-green fluorescent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Name</td>
			<td style="width:445px;">Company</td>
			<td style="width:167px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Software</td>
			<td style="width:445px;"></td>
			<td style="width:167px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Andor Solis</td>
			<td style="width:445px;">Andor Technology Ltd, Belfast, Northern Ireland</td>
			<td style="width:167px;"></td>
			<td>version 4.30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">LabVIEW</td>
			<td style="width:445px;">National Instruments, Austin, Texas, USA</td>
			<td style="width:167px;"></td>
			<td>version 2012, 32bit; misc. hardware control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">MDS control software</td>
			<td style="width:445px;">AA Opto-Electronic, Orsay, France</td>
			<td style="width:167px;"></td>
			<td>version 2.03a</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Coherent Connection</td>
			<td style="width:445px;">Coherent Inc, Santa Clara, CA, USA</td>
			<td style="width:167px;"></td>
			<td>version 3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Igor Pro</td>
			<td style="width:445px;">WaveMetrics Inc, Portland, OR, USA</td>
			<td style="width:167px;"></td>
			<td>version 6.37</td>
		</tr>
	</tbody>
</table>

using three-color single-molecule fret to study the correlation of protein interactions

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For many molecular machines in a cell proteins have to act together&#160;with interaction partners in a functional cycle to fulfill their task. The extension of two-color to multi-color smFRET makes it possible to simultaneously probe more than one interaction or conformational change. This not only adds a new dimension to smFRET experiments but it also offers the unique possibility to directly study the sequence of events and to detect correlated interactions when using an immobilized sample and a total internal reflection fluorescence microscope (TIRFM). Therefore, multi-color smFRET is a versatile tool for studying biomolecular complexes in a quantitative manner and in a previously unachievable detail.
Here, we demonstrate how to overcome the special challenges of multi-color smFRET experiments on proteins. We present detailed protocols for obtaining the data and for extracting kinetic information. This includes trace selection criteria, state separation, and the recovery of state trajectories from the noisy data using a 3D ensemble Hidden Markov Model (HMM). Compared to other methods, the kinetic information is not recovered from dwell time histograms but directly from the HMM. The maximum likelihood framework allows us to critically evaluate the kinetic model and to provide meaningful uncertainties for the rates.
By applying our method to the heat shock protein 90 (Hsp90), we are able to disentangle the nucleotide binding and the global conformational changes of the protein. This allows us to directly observe the cooperativity between the two nucleotide binding pockets of the Hsp90 dimer.

Multi-color smFRET measurements allow the direct detection of correlation between two or more distinct interaction sites. This renders the technique unique to investigate multi-component systems, such as protein complexes. We focus on the presentation of a three-color smFRET experiment here, which serves as an illustrative example.
The general workflow of the method is shown in Figure 1. The first p...

Watch this Scientific Journal Video about Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions at JoVE.com

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

Here, we present a protocol to obtain three-color smFRET data and its analysis with a 3D ensemble Hidden Markov Model. With this approach, scientists can extract kinetic information from complex protein systems, including cooperativity or correlated interactions.

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For ...

The overall goal of this procedure is to study the dynamics of complex protein systems in a quantitative way with a focus on correlated interactions such as cooperativity. This method can help answer key questions in the field of quantitative biology where protein complexes and their dynamic interactions are of central importance. The main advantage of this technique is that it allows dynamic multicolor single molecule FRET to be applied to biologically relevant systems.Prior to assembling the flow chamber cut a flow channel in a 40 micron thick transparent adhesive film spray quick fixing adhesive on the non adhesive side of the film and allow the adhesive to dry. To begin flow chamber assembly obtain a three millimeter thick PEG biotin PEG functionalized fused quartz slide with inlet and outlet holes. Apply the spray adhesive coated side of the ceiling film to the functionalized side of the slide aligning the channel with the inlet and outlet hooks.Heat the slide to 80 degrees Celsius on a hot plate for one minute. Press the film onto the slide for 30 seconds using an extra microscope slide to distribute the pressure evenly. And then allow the assembly to cool for one minute.Peel off the protective sheet covering the adhesive. Place a cover slip on the exposed adhesive to form the flow chamber. Heat the chamber to 80 degrees Celsius for one minute.Press the cover slip onto the adhesive for 30 seconds to seal the chamber and allow the assembled chamber to cool. Apply a drop of glycerol to the prism face before placing the flow chamber over the prism. Mount the flow chamber in a holder for a multi color prism type TIRF microscope.Connect the inlet and outlet tubing when finished. To begin configuring the instrument for the three color measurement open the electron multiplying CCD camera software set the EMCCD sensor to the lowest possible temperature. Set the camera vertical shift speed to 3.3 microseconds, choose normal vertical clock voltage, set the horizontal read out rate to 17 Millihertz at 16 bit, the pre-amplification gain to three and the electron multiplier gain level to 1000.Choose external acquisition triggering. Set the exposure time to 70 milliseconds. And the movie recording duration to 750 acquisition cycles.Create a new folder for the measurement files. Enable auto saving in the EMCCD software and set the movie frame file format to TIFF. Set the auto save file path to the newly created folder.Then open the software controlling the acousto-optical tunable filter, the laser control software, and the trigger software synchronizing the lasers the AOTF, the optical shutters, and the cameras. Use the AOTF to adjust the laser power to about three milliwatts before entering the prism. Load the triggering pattern that synchronizes all devices for the alternating laser excitation then mount the sample holder in the instrument.Place the end of the inlet tubing in a microcentrifuge tube containing the experiment buffer. Connect the outlet tubing to a syringe pump set to withdraw mode. Flush the chamber with 150 microliters of buffer.Focus the CCD camera on the functionalized interface align the excitation beams and bleach fluorescent contaminants. Then flow 300 microliters of a 0.25 milligram per milliliter solution of deglycosylated avidin and buffer into the chamber and incubate for one minute. Flush unbound deglycosylated avidin from the chamber with buffer.Then flow 300 microliters of buffer with a BSA concentration of 0.5 milligrams per milliliter through the chamber to block surface functionalization defects. Next, load the flow chamber with 150 microliter portions of biotinylated fluorescently labeled Hsp90 in BSA containing buffer at increasing Hsp90 concentrations until sufficient surface density is achieved. Wash unbound protein from the chamber with 300 microliters of BSA containing buffer.Load 150 microliters of a 25 nanomolar solution of labeled AMP-PMP and BSA containing buffer into the chamber and incubate for five minutes. Repeat the loading and incubation once. Then use a Piezo stepper to move the sample chamber perpendicular to the excitation beam to change the field of view as desired.Adjust the image focus if necessary. Start the camera recordings in the camera software by clicking the take signal button as shown. Initiate the excitation acquisition cycles in the trigger software to begin data acquisition.To begin data analysis, open the analysis software and import the recorded movies of the two cameras. Click find traces to determine the fluorescence intensity traces corresponding to potential single molecules. For each molecule, calculate the sum of all traces of that spot with the same excitation color.Evaluate the intensity profiles in all channels for a roughly flat plateau in the joint raw intensity and a single bleaching step for all excitation colors. Look for anti correlated behavior in the appropriate detection channels and the occurrence of red fluorescence in the trace. If all criteria are met save the fluorescence traces for further analysis.Only the indicated molecule fulfills the criteria in this example. Exclude traces that do not have flat plateaus, that show blinking events rather than anti correlation or that have multiple bleaching steps. Trace selection is a critical step in all single molecule techniques.Only traces that fulfill well defined criteria should be selected. Here these criteria are anti correlation, single bleaching steps, and flat plateaus. Next, display the saved molecules one after another and one set of intensity traces select a time interval in which all floor fours are already bleached.Calculate the beam background intensity for this time interval then select the FRET efficiency range in which both dyes on Hsp90 are present being sure to exclude traces with blinking events. Calculate the partial fluorescence traces. Exclude molecules with a low signal to noise ratio in the traces.Next, generate bind 2D projections of the partial fluorescence data and start the relative population calculation tool. Click Init. Draw a polygon around the peak of interest.And click count to calculate the relative population for that peak. Generate a 3D histogram of the partial fluorescence data and normalize the histogram. Excess the initial parameters for a 3D Gaussian fit and add the state populations to the end of the parameter vector.Fit the data and display the results. After defining the position and width of each state in the 3D partial fluorescent space initialize the Hidden Markov Model interface. Select the appropriate number of states, the number of dimensions of the input signal, and the input type.Optimize the HMM parameters to determine the maximum likelihood estimators for the transition probabilities. Repeat the population selection, fitting, and HMM optimization for data subsets. Finally calculate the confidence interval for the transition probabilities and collapse the conformational states to simplify the data for further analysis.The conformational states of the Hsp90 protein were studied with three color single molecule FRET in the presence of the labeled reporter nucleotide AMP-PMP. Five conformational states were distinguishable by fluorescence intensities with four of those states being functionally distinct. Three color single molecule FRET resulted in the partial fluorescence data spanning a 3D space.2D projections were used to help separate the states as all states theoretically expected under the experiment conditions are distinguishable by their partial florescence in 2D projections. The relative populations of these states were determined from the 2D projections and were used as constraints for 3D Gaussian fits. Subsequent ensemble 3D HMM optimization and modeling provided the extracted state transition probabilities.Confidence intervals were calculated for each extracted rate constant. Repeating the experiments in the presence of additional 250 micromolar unlabeled AMP-PMP elucidated the influence of the binding of one nucleotide on the second binding site in the Hsp90 dimer. Collapsing the bound and unbound conformations allowed the calculation of the average dwell time for the fluorescent AMP-PMP dissociation from Hsp90 in the presence and absence of additional unlabeled AMP-PMP.After watching this video you should have a good understanding of how to extract kinetic information from a dynamic protein system using multicolor FRET and a TIRF microscope. This technique paves the way for researchers in life sciences to determine correlated interactions in multi protein systems. This is important for a deeper understanding of fundamental regulatory mechanisms such as cooperativity.

using-three-color-single-molecule-fret-to-study-correlation-protein

Research

JoVE Journal

Biochemistry

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is necessary to characterize it at the structural and mechanistic level. Several biochemical and biophysical methods exist for such purpose. Among them, fluorescence anisotropy is a powerful technique particularly used when the fluorescence intensity of a fluorophore-labeled protein remains constant upon protein-protein interaction. In this technique, a fluorophore-labeled protein is excited with vertically polarized light of an appropriate wavelength that selectively excites a subset of the fluorophores according to their relative orientation with the incoming beam. The resulting emission also has a directionality whose relationship in the vertical and horizontal planes defines anisotropy (r) as follows: r=(IVV-IVH)/(IVV+2IVH), where IVV and IVH are the fluorescence intensities of the vertical and horizontal components, respectively. Fluorescence anisotropy is sensitive to the rotational diffusion of a fluorophore, namely the apparent molecular size of a fluorophore attached to a protein, which is altered upon protein-protein interaction. In the present text, the use of fluorescence anisotropy as a tool to study protein-protein interactions was exemplified to address the binding between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor like-1 GTPase (EFL1). Conventionally, labeling of a protein with a fluorophore is carried out on the thiol groups (cysteine) or in the amino groups (the N-terminal amine or lysine) of the protein. However, SBDS possesses several cysteines and lysines that did not allow site directed labeling of it. As an alternative technique, the dye 4&#39;,5&#39;-bis(1,3,2 dithioarsolan-2-yl) fluorescein was used to specifically label a tetracysteine motif, Cys-Cys-Pro-Gly-Cys-Cys, genetically engineered in the C-terminus of the recombinant SBDS protein. Fitting of the experimental data provided quantitative and mechanistic information on the binding mode between these proteins.

Protein interactions are at the heart of a cell&#39;s function. Calorimetric and spectroscopic techniques are commonly used to characterize them. Here we describe fluorescence anisotropy as a tool to study the interaction between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor-like 1 GTPase (EFL1).

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is ...

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. ...

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

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 ...

Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The modification presented includes the combination of this technique with cell-penetrating sequences. We propose to design cell-penetrating baits that can be incubated with living cells instead of cell lysates and therefore the interactions found will reflect those that occur within the intracellular context. Connexin43 (Cx43), a protein that forms gap junction channels and hemichannels is down-regulated in high-grade gliomas. The Cx43 region comprising amino acids 266-283 is responsible for the inhibition of the oncogenic activity of c-Src in glioma cells. Here we use TAT as the cell-penetrating sequence, biotin as the pull-down tag and the region of Cx43 comprised between amino acids 266-283 as the target to find intracellular interactions in the hard-to-transfect human glioma stem cells. One of the limitations of the proposed method is that the molecule used as bait could fail to fold properly and, consequently, the interactions found could not be associated with the effect. However, this method can be especially interesting for the interactions involved in signal transduction pathways because they are usually carried out by intrinsically disordered regions and, therefore, they do not require an ordered folding. In addition, one of the advantages of the proposed method is that the relevance of each residue on the interaction can be easily studied. This is a modular system; therefore, other cell-penetrating sequences, other tags, and other intracellular targets can be employed. Finally, the scope of this protocol is far beyond protein-protein interaction because this system can be applied to other bioactive cargoes such as RNA sequences, nanoparticles, viruses or any molecule that can be transduced with cell-penetrating sequences and fused to pull-down tags to study their intracellular mechanism of action.

This is a protocol to study intracellular protein-protein interactions based on the biotin-avidin pull-down system with the novelty of combining cell-penetrating sequences. The main advantage is that the target sequence is incubated with living cells instead of cell lysates and therefore the interactions will occur within the cellular context.

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

Fast oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting (HRPF) method used to study protein structure, protein-ligand interactions, and protein-protein interactions. FPOP utilizes a KrF excimer laser at 248 nm for photolysis of hydrogen peroxide to generate hydroxyl radicals which in turn oxidatively modify solvent-accessible amino acid side chains. Recently, we expanded the use of FPOP of in vivo oxidative labeling in Caenorhabditis elegans (C. elegans), entitled IV-FPOP. The transparent nematodes have been used as model systems for many human diseases. Structural studies in C. elegans by IV-FPOP is feasible because of the animal&#8217;s ability to uptake hydrogen peroxide, their transparency to laser irradiation at 248 nm, and the irreversible nature of the modification. The assembly of a microfluidic flow system for IV-FPOP labeling, IV-FPOP parameters, protein extraction, and LC-MS/MS optimized parameters are described herein.

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

In vivo fast photochemical oxidation of proteins (IV-FPOP) is a hydroxyl radical protein footprinting technique that allows for mapping of protein structure in their native environment. This protocol describes the assembly and set-up of the IV-FPOP microfluidic flow system.

Fast oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting (HRPF) method used to study protein structure, protein-ligand interactions, ...

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 ...

Optical Tweezers to Study RNA-Protein Interactions in Translation Regulation

RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the structure and function of an RNA can be modulated by various trans-acting factors, such as proteins, metabolites or other RNAs. Frameshifting RNA molecules, for instance, are regulatory RNAs located in coding regions, which direct translating ribosomes into an alternative open reading frame, and thereby act as gene switches. They may also adopt different folds after binding to proteins or other trans-factors. To dissect the role of RNA-binding proteins in translation and how they modulate RNA structure and stability, it is crucial to study the interplay and mechanical features of these RNA-protein complexes simultaneously.&#160;This work illustrates how to employ single-molecule-fluorescence-coupled optical tweezers to explore the conformational and thermodynamic landscape of RNA-protein complexes at a high resolution. As an example, the interaction of the SARS-CoV-2 programmed ribosomal frameshifting element with the trans-acting factor short isoform of zinc-finger antiviral protein is elaborated. In addition, fluorescence-labeled ribosomes were monitored using the confocal unit, which would ultimately enable the study of translation elongation. The fluorescence coupled OT assay can be widely applied to explore diverse RNA-protein complexes or trans-acting factors regulating translation and could facilitate studies of RNA-based gene regulation.

This protocol presents a complete experimental workflow for studying RNA-protein interactions using optical tweezers. Several possible experimental setups are outlined including the combination of optical tweezers with confocal microscopy.

RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the ...

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately 20 nm to accommodate large tRNA substrates at the A-, P- and E-sites. Consequently, the ribosome dynamics are naturally de-phased quickly. Single molecule method can detect each ribosome separately and distinguish inhomogeneous populations, which is essential to reveal the complicated mechanisms of multi-component systems. We report the details of a smFRET method based on the Nikon Ti2 inverted microscope to probe the ribosome dynamics between the ribosomal protein L27 and tRNAs. The L27 is labeled at its unique Cys 53 position and reconstituted into a ribosome that is engineered to lack L27. The tRNA is labeled at its elbow region. As the tRNA moves to different locations inside the ribosome during the elongation cycle, such as pre- and post- translocation, the FRET efficiencies and dynamics exhibit differences, which have suggested multiple subpopulations. These subpopulations are not detectable by ensemble methods. The TIRF-based smFRET microscope is built on a manual or motorized inverted microscope, with home-built laser illumination. The ribosome samples are purified by ultracentrifugation, loaded into a home-built multi-channel sample cell and then illuminated via an evanescent laser field. The reflection laser spot can be used to achieve feedback control of perfect focus. The fluorescence signals are separated by a motorized filter-turret and collected by two digital CMOS cameras. The intensities are retrieved via the NIS-Elements software.

Single molecule fluorescence energy transfer is a method that tracks the tRNA dynamics during ribosomal protein synthesis. By tracking individual ribosomes, inhomogeneous populations are identified, which shed light on mechanisms. This method can be used to track biological conformational changes in general to reveal dynamic-function relationships in many other complexed biosystems. Single molecule methods can observe non-rate limiting steps and low-populated key intermediates, which are not accessible by conventional ensemble methods due to the average effect.

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately ...