This work was supported by National Institutes of Health Grant GM094246, the American Heart Association Grant 11GRNT7890004, and the National Science Foundation Grant MCB-1110501.

1. Create the Construct Containing the Protein of Interest
Clone the gene expressing the protein of interest into a suitable vector. Use vectors such as the pcDNA series or pRK5 as they are well suited for expression in mammalian systems like HEK293 and CHO cells.
2. Select the Sites on the Protein to be Tagged with the Fluorophores
<ol>
	<li>Choose labeling sites that are able to reflect possible conformational changes within the protein. If possible, use a crystal structure of the protein or of a homologous protein to help make this determination. If there is no crystal structure available, use online software to predict the possible structure of the protein and hence receive insight into appropriate sites.</li>
	<li>Choose residues such that the side chains of the selected residues are surface exposed and accessible to the fluorophores so that the protein can be labeled.
	<ol>
		<li>Choose residues that are not critical to protein function, for example sites that are not involved in ligand binding.</li>
		<li>In introducing cysteine mutation, give preference to sites that are similar, for example serine, that would cause minimal perturbation to the protein structure.</li>
		<li>After choosing the labeling sites, make the mutations to ensure that the protein only gives LRET from these intended sites. Use standard site-directed mutagenesis protocols to mutate away non-disulfide-bonded cysteines that could potentially bind to maleimide-conjugated fluorescent tags. Do not mutate away disulfide-bonded cysteines and buried, free cysteines since they should not react with fluorescent dyes in the folded form of the protein.</li>
	</ol>
	</li>
	<li>Introduce cysteine residues in the desired sites by making point mutations using standard site-directed mutagenesis protocols.</li>
	<li>Include a protease cleavage site that can specifically cleave off one of the cysteines from the protein. If the protein sequence allows for it, introduce the site by conservatively mutating the protein to have a thrombin (recognition sequence LVPRGS) or Factor Xa (recognition sequence IDGR or IEGR) sequence near the introduced cysteine and accessible to protease cleavage; otherwise the tetra- or hexa-peptide sequence may be inserted as a whole. Choose the site such that upon cleavage one of the introduced cysteines dissociates from the rest of the protein&#8212;in certain cases, this may require two cleavage sites flanking a mutated cysteine.</li>
</ol>
3. Test the Expression and Functionality of the Protein
<ol>
	<li>Perform a western blot to confirm expression of the mutated protein.</li>
	<li>Perform a functional assay of the protein to ensure that the mutations have altered the protein function only minimally, if at all, to avoid studying conformational changes of a dysfunctional protein. 
	NOTE: Since all proteins have different functions, there is no single functional assay that is used specifically for LRET; however, some examples of functional assays include enzyme activity assays for enzymes, ligand-binding assays for receptors, and electrophysiology studies for ion channels.</li>
</ol>
4. Select the Fluorophores to be Used
Select fluorophores based on the expected distance range being measured such that the range is between 0.5-1x&#160;the R0 of the fluorophore pair. 
NOTE: This allows for an easier subtraction of the background, which typically has much longer lifetimes. For example, if the expected distance range being measured is around 35 &#197;, an appropriate fluorophore pair to use would be terbium chelate as the donor and Alexa 594 as the acceptor, because the R0 for this pair is 53 &#197; (Table 1).
5. Express the Protein by Transiently Transfecting the Required Amount of Mammalian Cells
Transiently transfect the protein of interest into the chosen mammalian cells using any of the common transfection reagents. Typically, use four 10-cm dishes per LRET experiment for HEK and CHO cell lines; however, this amount may vary depending on protein expression, stability, etc. Allow the cells to express the protein for 36-48 hr before harvesting.
6. Label the Proteins
<ol>
	<li>Detach cells from the culture dish. Detach HEK cells by simply pipetting buffer against the bottom of the dish. Detach CHO cells with a cell scraper, washing off the media with extracellular buffer.</li>
	<li>Collect the cells by centrifugation at 1,100 x g for 3 min. Use these same centrifuge settings for collecting cells after labeling, as well as after the subsequent washes.</li>
	<li>Suspend cells in 3 ml of extracellular buffer, then add donor and acceptor fluorophores in equimolar amounts up to a final concentration of 100-300 nM. Incubate on a rotator for 1 hr at RT.</li>
	<li>Wash these labeled cells 3-4x&#160;with extracellular buffer to remove unbound fluorophores, then suspend these cells in extracellular buffer (usually 2 ml) for LRET measurements.</li>
	<li>As a control, label a separate set of cells with only donor fluorophore (terbium chelate) without addition of any acceptor fluorophore. 
	NOTE: Data from these donor-only experiments is necessary to complete analysis. These experiments can be done on the same or different days.</li>
	<li>Again, perform functional validation, this time with labeled mutant protein, as the labeling process may also interfere with function depending on the site used for labeling. 
	NOTE: These experiments can be done on the same or different days.</li>
</ol>
7. Set up the LRET Experiment
<ol>
	<li>Place the resuspended cells in a quartz cuvette with a minimum volume of 1 ml.</li>
	<li>Turn on the computer and the instrument and adjust the parameters of the data acquisition program accordingly.
	<ol>
		<li>Set the excitation wavelength to the absorbance range of the donor fluorophore (330-340 nm works well for terbium chelate).</li>
		<li>Set the emission wavelength appropriately, keeping in mind that the acceptor emission varies based on the acceptor used. Importantly, select a detection wavelength that measures only the acceptor emission and does not include any bleed-through from the donor&#8217;s emission. For example, use a wavelength of 565 nm for Alexa 555 as an acceptor Table 1. For donor-only measurements, in which protein was labeled only with donor but without acceptor, use a wavelength of 545 nm for measuring terbium chelate emission.</li>
		<li>Set the length of emission detection to be at least three times the expected LRET lifetime to ensure that a long-lifetime component will not be missed.</li>
	</ol>
	</li>
	<li>Perform the scan. Do at least three scans of 99 sweeps to ensure consistency of results. Save the results as a text file (*.txt).</li>
	<li>To measure conformational changes of a protein with regard to different conditions (such as the addition of a ligand), alter those conditions and again perform at least three scans of 99 sweeps each on the same sample under this new condition. If studying the effects of glutamate on the conformation of glutamate receptors, for example, add glutamate to 1 mM to the same sample scanned in step 7.3, and then scan again.</li>
	<li>Add up to five units of the appropriate protease and take scans continually until cleavage is complete and no further change is seen in the lifetime for three successive scans. Usually, cleavage is complete in two to 3 hr. If a Factor Xa cleavage site is being used for protease cleavage, for example, add 3 &#181;l of Factor Xa to the sample, and scan every 30 min for 3 hr.</li>
</ol>
8. Analyze the Data Obtained
<ol>
	<li>Open the data analysis software.</li>
	<li>Load the fluorescence lifetime data by using Import ASCII to open the text files. Load all the replicates as well as the final background measurements, into one file.</li>
	<li>Average the data from all the scans for each experimental condition to get the final data. To do so, highlight the columns that contain the individual trials, then under the Data menu in the menu bar, click on Statistics on Rows to display the average fluorescence intensities from the trials, as well as the standard deviation and standard error.</li>
	<li>Plot the average values as a line graph with fluorescence intensity on the Y-axis and lifetime in microseconds on the X-axis to create a curve that represents the lifetime of the sensitized emission of the acceptor. Change the Y-axis of the plot to a log scale, to allow for better visualization of the data.</li>
	<li>Repeat step 8.3 on the background measurement data, averaging the final scans that show overlap indicating complete cleavage.</li>
	<li>Display the mean background data on the same plot that contains the averaged raw data. To do so, open the Layer Control dialog box found under the Plot menu. Then, transfer the data containing the background mean into the list of Layer Contents.</li>
	<li>Align the mean background data to the mean raw data. To do so, use the Simple Math function under the Math menu to multiply or divide the background data as needed until the tail end of the background overlaps with the tail end of the raw data. 
	NOTE: At this tail end, the lifetime from the protein of interest should have already completely decayed away; what is aligned is simply the background signal present both before and after protease cleavage.</li>
	<li>Subtract the aligned background signal from the initial raw LRET signal, again using the Simple Math function.</li>
	<li>Fit the data to an exponential decay (single or multiple exponentials, depending on the sites and the experimental design).
	<ol>
		<li>Set the start boundary for fitting to begin after the end of the laser pulse. Use this same startpoint for all subsequent curve fittings.</li>
		<li>In the Select Fitting Function dialog box, select exponential decay so as to fit the lifetime to the following equation for a single exponential decay: 
		<img alt="Equation 4" fo:content-width="2in" src="/files/ftp_upload/51895/51895eq4.jpg" width="200" />&#160;Eq. 4 
		where, y represents the fluorescence intensity, y0 represents the background intensity due to noise from the system, A1 is the signal intensity, t is the fluorescence lifetime, x is the time, and x0 is the time offset.</li>
		<li>Fix x0 to 0, start the Fitting Function, and fit the data. 
		NOTE: If the experiment is designed to have signal only from one pair of sites, the resulting data should easily be fit by a single exponential decay8. The residual of the fit is a good method to determine the goodness of the fit and if additional decay lifetimes are required.</li>
	</ol>
	</li>
	<li>Use the lifetimes obtained from the data to calculate the distance between fluorophores using the F&#246;rster equation (a rearrangement of Equations 1 and 2 above): 
	<img alt="Equation 5" fo:content-width="2in" src="/files/ftp_upload/51895/51895eq5.jpg" width="200" />&#160;Eq. 5 
	NOTE: All the variables are as mentioned above with &#964;DA having been measured as the sensitized acceptor emission lifetime. Further details and examples on measurements of R0 and R can be found elsewhere7,9,10.</li>
	<li>Repeat the experiment and data analysis at least three times to ensure reproducibility. Inconsistency between individual repetitions of the same measurement calls into question the validity of the data; use additional control experiments or repetitions. Calculate errors in measurement using the free Gustavus Error analysis calculator (developed by Dr. Thomas Huber) or a similar system that propagates the error in the fits of the lifetime.</li>
</ol>

<ol>
	<li>Selvin, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+and+biophysical+applications+of+lanthanide-based+probes.">Principles and biophysical applications of lanthanide-based probes.</a> Annual Review of Biophysics and Biomolecular Structure. 31, 275-302 (2002).</li><li>Stryer, L., Haugland, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+transfer:+a+spectroscopic+ruler.">Energy transfer: a spectroscopic ruler.</a> Proceedings of the National Academy of Sciences of the United States of America. 58, 719-726 (1967).</li><li>Stryer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+energy+transfer+as+a+spectroscopic+ruler.">Fluorescence energy transfer as a spectroscopic ruler.</a> Annual Review of Biochemistry. 47, 819-846 (1978).</li><li>Selvin, P. R., Hearst, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Luminescence+energy+transfer+using+a+terbium+chelate:+improvements+on+fluorescence+energy+transfer.">Luminescence energy transfer using a terbium chelate: improvements on fluorescence energy transfer.</a> Proceedings of the National Academy of Sciences of the United States of America. 91, 10024-10028 (1994).</li><li>Chen, J., Selvin, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thiol-reactive+luminescent+chelates+of+terbium+and+europium.">Thiol-reactive luminescent chelates of terbium and europium.</a> Bioconjugate Chemistry. 10, 311-315 (1999).</li><li>Ge, P., Selvin, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thiol-reactive+luminescent+lanthanide+chelates:+part+2.">Thiol-reactive luminescent lanthanide chelates: part 2.</a> Bioconjugate Chemistry. 14, 870-876 (2003).</li><li>Gonzalez, J., Rambhadran, A., Du, M., Jayaraman, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LRET+investigations+of+conformational+changes+in+the+ligand+binding+domain+of+a+functional+AMPA+receptor.">LRET investigations of conformational changes in the ligand binding domain of a functional AMPA receptor.</a> Biochemistry. 47, 10027-10032 (2008).</li><li>Ramaswamy, S. S., Maclean, D. M., Gorfe, A. A., Jayaraman, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proton+mediated+conformational+changes+in+an+Acid+Sensing+Ion+Channel.">Proton mediated conformational changes in an Acid Sensing Ion Channel.</a> J. Bio. Chem. 288, (2013).</li><li>Rambhadran, A., Gonzalez, J., Jayaraman, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+changes+at+the+agonist+binding+domain+of+the+N-methyl-D-aspartic+acid+receptor.">Conformational changes at the agonist binding domain of the N-methyl-D-aspartic acid receptor.</a> The Journal of Biological Chemistry. 286, 16953-16957 (2011).</li><li>Rambhadran, A., Gonzalez, J., Jayaraman, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subunit+arrangement+in+N-methyl-D-aspartate+(NMDA)+receptors.">Subunit arrangement in N-methyl-D-aspartate (NMDA) receptors.</a> The Journal of Biological Chemistry. 285, 15296-15301 (2010).</li><li>Kokko, T., Kokko, L., Soukka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Terbium(III)+chelate+as+an+efficient+donor+for+multiple-wavelength+fluorescent+acceptors.">Terbium(III) chelate as an efficient donor for multiple-wavelength fluorescent acceptors.</a> Journal of Fluorescence. 19, 159-164 (2009).</li></ol>

The authors have nothing to disclose.

LRET is a powerful technique that allows scientists to measure distances between domains within a single protein as well as between subunits in a multimeric protein. As such, LRET is well-suited to examining the conformational changes and dynamics of proteins or other macromolecules. The above protocol should allow the properly equipped lab to easily test their hypotheses; however, there are many common sources of error that may plague the new investigator. If little or no LRET signal is seen, first check the wavelength ...

Luminescence Resonance Energy Transfer (LRET) is a derivative of the well-known Fluorescence Resonance Energy Transfer (FRET) technique1. Similar to FRET, LRET can be used to measure distances and distance changes between donor and acceptor fluorophores attached to specific sites on the protein of interest within the range of 10-100 &#197;1-3. The principles of LRET are also similar to FRET in that resonance energy transfer occurs between two proximal fluorophores when the emission spectrum of the donor fluorophore overlaps with the absorption spectrum of the acceptor fluorophore. The efficiency of this transfer is related to the distance between the two fluorophores by the following equation:
<img alt="Equation 1" fo:content-width="1.2in" src="/files/ftp_upload/51895/51895eq1.jpg" width="120" />&#160;Eq. 1
where R is the distance between the two fluorophores, E is the efficiency of energy transfer, and R0, discussed below, is the F&#246;rster radius for the fluorophore pair, i.e. the distance at which efficiency of transfer is half-maximal. From this equation, one can see that efficiency is related to the magnitude of the distance raised to the inverse sixth power1. It is this inverse sixth power dependence that allows for FRET and LRET measurements to be exquisitely sensitive even to small distance changes when near the R0 of the FRET pair. The ability to specifically label desired sites on proteins or other macromolecules allows one to take advantage of this sensitivity to monitor conformational changes.
When compared to FRET, which uses conventional organic dye molecules, LRET offers additional advantages. In LRET, instead of using an organic dye as the donor fluorophore, a lanthanide series cation, typically Tb3+ or Eu3+, is used1,4-6. Fluorophores that fall under this category, e.g., terbium chelate, are also very versatile in that they can be used with a wide range of acceptor fluorophores. This flexibility is made possible because the emission spectra of chelated lanthanides contain multiple sharp emission peaks, allowing for a single species of donor fluorophore to be used with one of a wide variety of acceptor fluorophores. Thus, sensitized acceptor emission can be detected without any fear of contaminating bleed-through from donor emission5. The experimenter selects the specific acceptor based on the expected distance between the two fluorophores (Figure 1 and Table 1). In these chelated lanthanide fluorophores, the metal ion is chelated by a molecule that contains an antenna group that sensitizes the normally poorly-absorbing lanthanide to excitation as well as a bioreactive functional group to tether the ion to a specific functional group on the macromolecule1,5,6. Once excited, lanthanides relax to the ground state via the release of photons with a decay rate in the millisecond range. Because the decay is neither a singlet-to-singlet relaxation nor a triplet-to-singlet relaxation, the emission of photons cannot properly be called fluorescence or phosphorescence, but is more properly termed luminescence1. The long decay of lanthanide luminescence greatly helps in lifetime measurements. Lifetime measurements can then be used to determine efficiency via the following relation:
<img alt="Equation 2" fo:content-width="1.2in" src="/files/ftp_upload/51895/51895eq2.jpg" width="120" />&#160;Eq. 2
where, E is the efficiency of transfer, &#964;D is the lifetime of the donor (chelated lanthanide) when not participating in energy transfer, and &#964;DA is the lifetime of the donor when participating in energy transfer with the acceptor. With LRET, &#964;DA can also be measured as the lifetime of the sensitized acceptor emission because terbium&#8217;s lifetime is so much greater than an organic acceptor fluorophore. The acceptor emits with the same lifetime as its inciting excitation (donor lanthanide), and any contribution to the lifetime from the acceptor&#8217;s own intrinsic fluorescence lifetime is relatively negligible. By measuring the sensitized emission rather than donor emission, we also eliminate the need to ensure labeling at exactly a 1:1 ratio of donor to acceptor. Protein can instead be labeled simultaneously with both acceptor and donor fluorophores. A heterogeneously labeled population will result, but double-donor labeled proteins will not emit in the acceptor wavelength and double-acceptor labeled proteins will not be excited. Moreover, the distance between fluorophores should be the same, regardless of which cysteine site a given fluorophore attaches to, especially when using the isotropic lanthanides as a donor, so the need to specify a given site to receive either the donor or acceptor is unnecessary. Intensity may be affected with a heterogeneous population, but should still be more than sufficient to be detected.
When planning experiments, the choice of fluorophores should be dictated by the R0 value of the pair as well as the expected distance range being measured. The R0 value is defined by the following equation:
<img alt="Equation 3" fo:content-width="2.5in" src="/files/ftp_upload/51895/51895eq3.jpg" width="250" />&#160;Eq. 3
where, R0&#160;is the F&#246;rster radius in Angstroms, &#954;2 is the orientation factor between the two dyes (usually assumed to be 2/3), &#981;D is the quantum yield of the donor, J is the spectral overlap integral between the donor&#39;s emission spectrum and the acceptor&#39;s absorbance spectrum in M-1cm-1nm4, and n is the refractive index of the medium1.
Our laboratory has added a modification to the conventional LRET technique by introducing a protease recognition site between the donor and acceptor label sites on the protein being probed. This modification allows for investigation in non-purified systems such as whole mammalian cells7. This technique is particularly useful when using cysteines as sites for labeling, since in the process of labeling with maleimide-conjugated dyes that bind to cysteine sulfhydryl groups, other proteins on the cells that have cysteines are also labeled. However, by including protease cleavage sites on the protein of interest and measuring lifetimes before and after cleavage, the experimenter can quantitatively subtract the background signal after protease cleavage from the raw signal. This subtraction isolates the specific signal arising from the protein of interest (Figure 2). Using the modification described above, LRET can be used to measure distance changes between the terbium chelate donor and the acceptor probe on a protein, and thus monitor conformational changes in the protein&#8217;s near physiological state without the requirement for purification.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51895/51895fig1highres.jpg" width="500" /> 
Figure 1.The absorption and emission spectra of chelated terbium in black, as well as a representative acceptor, Alexa 488, in red. Notice the multiple emission peaks and the sharp, narrow emission range for each peak of terbium chelate. This pattern allows for terbium to be used with a variety of acceptor fluorophores and facilitates the measurement of sensitized emission within those ranges where terbium shows no emission. Terbium&#8217;s emission peak at 486 nm overlaps quite well with the absorption peak of Alexa 488, allowing for resonance energy transfer to occur between the two fluorophores. A wavelength of 515 nm is an excellent choice to detect sensitized emission for this pair as it is in the valley between the terbium emission peaks, and quite near Alexa 488&#8217;s emission peak of 520 nm. Note that being near the acceptor peak, though desirable, is not required&#8212;565 nm is still able to detect Alexa 488 emission without also detecting terbium emission.
<table border="0" cellpadding="0" cellspacing="0" width="374">
	<tbody>
		<tr height="27">
			<td height="27" style="height:27px;width:147px;">Acceptor Fluorophore</td>
			<td style="width:45px;">R0 (&#197;)</td>
			<td style="width:183px;">Emission wavelength (nm)</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:147px;">Atto 465</td>
			<td align="right" style="width:45px;">36</td>
			<td align="right" style="width:183px;">508</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:147px;">Fluorescein</td>
			<td align="right" style="width:45px;">45</td>
			<td align="right" style="width:183px;">515</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:147px;">Alexa 488</td>
			<td align="right" style="width:45px;">46</td>
			<td align="right" style="width:183px;">515</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:147px;">Alexa 680</td>
			<td align="right" style="width:45px;">52</td>
			<td align="right" style="width:183px;">700</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:147px;">Alexa 594</td>
			<td align="right" style="width:45px;">53</td>
			<td align="right" style="width:183px;">630</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:147px;">Alexa 555</td>
			<td align="right" style="width:45px;">65</td>
			<td align="right" style="width:183px;">565</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:147px;">Cy3</td>
			<td align="right" style="width:45px;">65</td>
			<td align="right" style="width:183px;">575</td>
		</tr>
	</tbody>
</table>
Table 1. A list of commonly used acceptor fluorophores for LRET using terbium chelate as the donor11. The R0 values were measured when the donor and acceptor were attached to the soluble agonist binding domain of AMPA receptors. It is ideal to measure the R0 value again for each new system being studied.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51895/51895fig2highres.jpg" width="500" /> 
Figure 2. An overview of the LRET method presented. (A) The AMPA receptor is a membrane protein which undergoes conformational changes upon ligand-binding. The clamshell-shaped ligand-binding domain is circled here in red. (B) The ligand-binding domain of AMPA when not bound to protein exists in an open conformation (left). When bound to ligand glutamate, the protein closes around its ligand (right). By placing fluorophores at probative sites on the LBD, the nature of this conformational change can be seen as the distance between the fluorophores changes, which will then affect fluorescence lifetime. (C) When labeling whole cells, labeling of both the protein of interest as well as background membrane proteins may occur (left). After protease cleavage, LRET signal from the protein of interest will disappear due to the release of a soluble fragment, leaving background signal intact (right). This background signal can then be subtracted from the raw signal.

<table border="0" cellpadding="0" cellspacing="0" width="1394">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Name of Reagent/ Equipment</td>
			<td style="width:223px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:693px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">LanthaScreen Thiol Reactive Tb Chelate</td>
			<td style="width:223px;">Life Technologies</td>
			<td style="width:119px;">PV3579</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Acceptor fluorophore-Fluorescein-5-Maleimide</td>
			<td style="width:223px;">Life Technologies&#160;</td>
			<td style="width:119px;">F-150</td>
			<td>Choice of acceptor depends on the specific experiment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Acceptor fluorophore-Alexa Fluor 488 C5 Maleimide</td>
			<td style="width:223px;">Life Technologies&#160;</td>
			<td style="width:119px;">A-10254</td>
			<td>Choice of acceptor depends on the specific experiment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Acceptor fluorophore-Alexa Fluor 555 C2 Maleimide</td>
			<td style="width:223px;">Life Technologies&#160;</td>
			<td style="width:119px;">A-20346</td>
			<td>Choice of acceptor depends on the specific experiment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Acceptor fluorophore-Alexa Fluor 594 C5 Maleimide</td>
			<td style="width:223px;">Life Technologies&#160;</td>
			<td style="width:119px;">A-10256</td>
			<td>Choice of acceptor depends on the specific experiment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Acceptor fluorophore-Alexa Fluor 680 C2 Maleimide</td>
			<td style="width:223px;">Life Technologies&#160;</td>
			<td style="width:119px;">A-20344</td>
			<td>Choice of acceptor depends on the specific experiment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">QuantaMaster 3-SS</td>
			<td style="width:223px;">Photon Technology International</td>
			<td style="width:119px;"></td>
			<td>Spectrofluorometer should have pulsed excitation with the ability to measure lifetimes in the ms range</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">FluoreScan 2.0</td>
			<td style="width:223px;">Photon Technology International</td>
			<td style="width:119px;"></td>
			<td>Data Acquisition Software used in manuscript. Software provided with fluorescence instrument.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Origin 8.6</td>
			<td style="width:223px;">OriginLab</td>
			<td style="width:119px;"></td>
			<td>Origin is the data analysis software used in protocol. Can use other similar data analysis software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Quartz Cuvette</td>
			<td style="width:223px;">Starna Cells, Inc</td>
			<td style="width:119px;">3-Q-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Stir Bar&#160;</td>
			<td style="width:223px;">Bel-Art Products</td>
			<td style="width:119px;">F37119-0007</td>
			<td>Used in cuvette to keep cells in suspension. Can use any stir bar that fits the cuvette</td>
		</tr>
	</tbody>
</table>

luminescence resonance energy transfer to study conformational changes in membrane proteins expressed in mammalian cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

A successful LRET measurement with a lanthanide donor should have a donor only lifetime in the millisecond time range. The lifetime of sensitized LRET emission for the protein labeled with both the donor and the acceptor will be notably shorter, with a lifetime in the microsecond range after background subtraction Figure 3. Protease cleavage results in an increase in lifetime that should become stable over time (i.e. no longer changing), showing that the protease cleavage is complete Fig...

Watch this Scientific Journal Video about Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells at JoVE.com

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

luminescence-resonance-energy-transfer-to-study-conformational

Research

JoVE Journal

Bioengineering

11.9K Views.  University of Texas Health Science Center at Houston. We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

Video: Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural biology 1. In recent years, this method has achieved great success in studying structures of proteins and macromolecular complexes 2, 3. Compared with electron cryomicroscopy (cryoEM), in which frozen hydrated protein samples are embedded in a thin layer of vitreous ice 4, negative staining is a simpler sample preparation method in which protein samples are embedded in a thin layer of dried heavy metal salt to increase specimen contrast 5. The enhanced contrast of negative stain EM allows examination of relatively small biological samples. In addition to determining three-dimensional (3D) structure of purified proteins or protein complexes 6, this method can be used for much broader purposes. For example, negative stain EM can be easily used to visualize purified protein samples, obtaining information such as homogeneity/heterogeneity of the sample, formation of protein complexes or large assemblies, or simply to evaluate the quality of a protein preparation.
In this video article, we present a complete protocol for using an EM to observe negatively stained protein sample, from preparing carbon coated grids for negative stain EM to acquiring images of negatively stained sample in an electron microscope operated at 120kV accelerating voltage. These protocols have been used in our laboratory routinely and can be easily followed by novice users.

Visualizing protein samples by negative stain electron microscopy (EM) has become a popular structural analysis method. It is useful for quantitative structural analysis, such as calculating a 3D reconstruction of the molecules being studied, and also for qualitative examination of the quality of protein preparations. In this article we present detailed protocols for preparing the EM grids, staining the sample and visualizing the sample in an electron microscope. Novice users can follow these protocols easily and to utilize negative stain EM as a routine assay, in addition to other biochemical assays, for evaluating their protein samples.

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural ...

An Experimental System to Study Mechanotransduction in Fetal Lung Cells

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key component of lung development is the differentiation of alveolar type II epithelial cells, the major source of pulmonary surfactant. These cells also participate in fluid homeostasis in the alveolar lumen, host defense, and injury repair. In addition, distal lung parenchyma cells can be directly exposed to exaggerated stretch during mechanical ventilation after birth. However, the precise molecular and cellular mechanisms by which lung cells sense mechanical stimuli to influence lung development and to promote lung injury are not completely understood. Here, we provide a simple and high purity method to isolate type II cells and fibroblasts from rodent fetal lungs. Then, we describe an in vitro system, The Flexcell Strain Unit, to provide mechanical stimulation to fetal cells, simulating mechanical forces in fetal lung development or lung injury. This experimental system provides an excellent tool to investigate molecular and cellular mechanisms in fetal lung cells exposed to stretch. Using this approach, our laboratory has identified several receptors and signaling proteins that participate in mechanotransduction in fetal lung development and lung injury.

Mechanical forces play a key role in lung development and lung injury. Here, we describe a method to isolate rodent fetal lung type II epithelial cells and fibroblasts and to expose them to mechanical stimulation using an in vitro system.

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key ...

Quantitative FRET (F&#246;rster Resonance Energy Transfer) Analysis for SENP1 Protease Kinetics Determination

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein activity and have diverse roles in many biological processes. For example, SUMO covalently modifies a large number or proteins with important roles in many cellular processes, including cell-cycle regulation, cell survival and death, DNA damage response, and stress response 1-5. SENP, as SUMO-specific protease, functions as an endopeptidase in the maturation of SUMO precursors or as an isopeptidase to remove SUMO from its target proteins and refresh the SUMOylation cycle 1,3,6,7.
The catalytic efficiency or specificity of an enzyme is best characterized by the ratio of the kinetic constants, kcat/KM. In several studies, the kinetic parameters of SUMO-SENP pairs have been determined by various methods, including polyacrylamide gel-based western-blot, radioactive-labeled substrate, fluorescent compound or protein labeled substrate 8-13. However, the polyacrylamide-gel-based techniques, which used the "native" proteins but are laborious and technically demanding, that do not readily lend themselves to detailed quantitative analysis. The obtained kcat/KM from studies using tetrapeptides or proteins with an ACC (7-amino-4-carbamoylmetylcoumarin) or AMC (7-amino-4-methylcoumarin) fluorophore were either up to two orders of magnitude lower than the natural substrates or cannot clearly differentiate the iso- and endopeptidase activities of SENPs.
Recently, FRET-based protease assays were used to study the deubiquitinating enzymes (DUBs) or SENPs with the FRET pair of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) 9,10,14,15. The ratio of acceptor emission to donor emission was used as the quantitative parameter for FRET signal monitor for protease activity determination. However, this method ignored signal cross-contaminations at the acceptor and donor emission wavelengths by acceptor and donor self-fluorescence and thus was not accurate.
We developed a novel highly sensitive and quantitative FRET-based protease assay for determining the kinetic parameters of pre-SUMO1 maturation by SENP1. An engineered FRET pair CyPet and YPet with significantly improved FRET efficiency and fluorescence quantum yield, were used to generate the CyPet-(pre-SUMO1)-YPet substrate 16. We differentiated and quantified absolute fluorescence signals contributed by the donor and acceptor and FRET at the acceptor and emission wavelengths, respectively. The value of kcat/KM was obtained as (3.2 &plusmn; 0.55) x107 M-1s-1 of SENP1 toward pre-SUMO1, which is in agreement with general enzymatic kinetic parameters. Therefore, this methodology is valid and can be used as a general approach to characterize other proteases as well.

Quantitative FRET F&amp;#246;rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination

A novel method involving quantitative analysis of FRET (F&ouml;rster Resonance Energy Transfer) signals is described for studying enzyme kinetics. KM and kcat were obtained for the hydrolysis of the catalytic domain of SENP1 (SUMO/Sentrin specific protease 1) to pre-SUMO1 (Small Ubiquitin-like MOdifier). The general principles of this quantitative-FRET-based protease kinetic study can be applied to other proteases.

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein ...

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to identify and characterize their interactions are necessary5. To this end, we developed the Membrane Strep-protein interaction experiment, called Membrane-SPINE6. This technique combines in vivo cross-linking using the reversible cross-linker formaldehyde with affinity purification of a Strep-tagged membrane bait protein. During the procedure, cross-linked prey proteins are co-purified with the membrane bait protein and subsequently separated by boiling. Hence, two major tasks can be executed when analyzing protein-protein interactions (PPIs) of membrane proteins using Membrane-SPINE: first, the confirmation of a proposed interaction partner by immunoblotting, and second, the identification of new interaction partners by mass spectrometry analysis. Moreover, even low affinity, transient PPIs are detectable by this technique. Finally, Membrane-SPINE is adaptable to almost any cell type, making it applicable as a powerful screening tool to identify PPIs of membrane proteins.

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

A biochemical approach is described to identify in vivo protein-protein interactions (PPI) of membrane proteins. The method combines protein cross-linking, affinity purification and mass spectrometry, and is adaptable to almost any cell type or organism. With this approach, even the identification of transient PPIs becomes possible.

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to ...

A Protocol for Using F&#246;rster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. Nesprin-2G is a key component of the LINC complex that connects the actin cytoskeleton to membrane proteins (SUN domain proteins) in the perinuclear space. These membrane proteins connect to lamins inside the nucleus. Recently, a F&#246;rster Resonance Energy Transfer (FRET)-force probe was cloned into mini-Nesprin-2G (Nesprin-TS (tension sensor)) and used to measure tension across Nesprin-2G in live NIH3T3 fibroblasts. This paper describes the process of using Nesprin-TS to measure LINC complex forces in NIH3T3 fibroblasts. To extract FRET information from Nesprin-TS, an outline of how to spectrally unmix raw spectral images into acceptor and donor fluorescent channels is also presented. Using open-source software (ImageJ), images are pre-processed and transformed into ratiometric images. Finally, FRET data of Nesprin-TS is presented, along with strategies for how to compare data across different experimental groups.

A Protocol for Using F&amp;#246;rster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

A number of FRET-based force biosensors have recently been developed, enabling the protein-specific resolution of intracellular force. In this protocol, we demonstrate how one of these sensors, designed for the linker of the nucleoskeleton-cytoskeleton (LINC) complex protein Nesprin-2G can be used to measure actomyosin forces on the nuclear LINC complex.

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

Melt Electrospinning Writing of Three-dimensional Poly(&amp;#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based method is one of the simplest techniques for the mass production of cellular aggregates, but it does not protect the cultures against over-aggregation, which occurs when they gather at the bottom center of the culture vessel. To solve this problem, we developed an O-shaped dish and an O-shaped bag, neither of which contains a central region. Aggregates grown in either O-shaped culture vessel were noticeably more uniform in size than aggregates grown in conventional vessels. Histological analyses showed that aggregates in conventional culture dishes contained necrotic cores most likely caused by a poor oxygen supply. In contrast, aggregates that were grown in the O-shaped bag, even those with similar diameters to aggregates in conventional culture dishes, did not show necrotic cores. These results suggest that the O-shaped bag provides sufficient oxygen to the aggregates due to the oxygen permeability of the bag material. We, therefore, propose that this novel gas-permeable O-shaped culture bag is suitable for the mass production of uniform aggregates that are necessary in various biotechnological fields.

Here we present a protocol for using O-shaped vessels, specialized for suspension cultures of cellular aggregates, with orbital shaking. The HEK293 cells grown in this bag form more homogeneous aggregates than those grown in conventional culture vessels.

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based ...

Characterizing Single-Molecule Conformational Changes Under Shear Flow with Fluorescence Microscopy

Single-molecule behavior under mechanical perturbation has been characterized widely to understand many biological processes. However, methods such as atomic force microscopy have limited temporal resolution, while F&#246;rster resonance energy transfer (FRET) only allow conformations to be inferred. Fluorescence microscopy, on the other hand, allows real-time in situ visualization of single molecules in various flow conditions. Our protocol describes the steps to capture conformational changes of single biomolecules under different shear flow environments using fluorescence microscopy. The shear flow is created inside microfluidic channels and controlled by a syringe pump. As demonstrations of the method, von Willebrand factor (VWF) and lambda DNA are labeled with biotin and fluorophore and then immobilized on the channel surface. Their conformations are continuously monitored under variable shear flow using total internal reflection (TIRF) and confocal fluorescence microscopy. The reversible unraveling dynamics of VWF are useful for understanding how its function is regulated in human blood, while the conformation of lambda DNA offers insights into the biophysics of macromolecules. The protocol can also be widely applied to study the behavior of polymers, especially biopolymers, in varying flow conditions and to investigate the rheology of complex fluids.

We present a protocol for immobilizing single macromolecules in microfluidic devices and quantifying changes in their conformations under shear flow. This protocol is useful for characterizing the biomechanical and functional properties of biomolecules such as proteins and DNA in a flow environment.

Single-molecule behavior under mechanical perturbation has been characterized widely to understand many biological processes. However, methods such as ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...