Name: fluorescence anisotropy as a tool to study protein-protein interactions
Uploaded: 2016-10-21T15:00:00
Description: Watch this Scientific Journal Video about Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions at JoVE.com

Authors acknowledge the financial support from CONACyT project numbers 167359 and 177138, and from DGAPA-UNAM project number IN201615.

1. SBDS-FlAsH Tag Protein Expression and Purification
NOTE: For the anisotropy experiments, a FlAsH-tag corresponding to the sequence Cys-Cys-Pro-Gly-Cys-Cys was added to the C-terminus of the human SBDS coding sequence by PCR. This construct was subcloned into the expression vector pRSET-A and transformed into Escherichia coli C41 cells to express a protein encoding a N-terminal hexahistidine tag (His-tag), the human SBDS coding sequence and a C-terminus FlAsH tag 10.
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	<li>SBDS-...

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Most biochemical experiments with proteins require not only pure protein but also large amounts of them, irrespective of the technique used. For this reason, the proteins used for this type of experiments are obtained by heterologous expression, as it was the case presented here. Florescence spectroscopy requires the presence of a fluorophore in the studied molecule. Aromatic residues constitute the intrinsic fluorophores of a protein, however, using their signal to study protein-protein interactions complicates the anal...

Cells contain a multitude of biomacromolecules that constantly interact with each other. This association gives rise to complexes that participate in the cellular pathways responsible for their functioning in signal transduction, regulation of gene expression and cell migration amongst others. All protein-protein interactions that occur in a cell comprise a network known as the interactome. In Saccharomyces cerevisiae more than 70% of its proteins have been shown to have interacting partners 1. Understanding the interactome of a cell and their functions provide relevant information on the complexity and diversity of living organisms. Several methodologies have been described to identify and characterize protein-protein interactions. Different high through put methods such as yeast two-hybrid 2, protein-fragment complementation assays 3, affinity purification 4 coupled to mass spectrometry and protein microarrays are used to identify an interaction 5,6. Once identified, it is necessary to validate it and this may vary on a case-by-case basis. Typically, these experiments involve disrupting the interaction itself at the level of the individual members of the interaction pair, e.g., by gene deletion or overexpression of one of the proteins, and then looking for changes in the properties or function of the other member at the cellular level. Subsequently, biophysical techniques 7 are used to characterize the protein-protein interaction at the molecular level. To this end, the structure of protein complexes are determined by X-ray crystallography, nuclear magnetic resonance and cryo-electron microscopy while calorimetry and fluorescence spectroscopy are used to quantitatively and mechanistically describe them.
In this work, fluorescence anisotropy was used as a technique to characterize the interaction between the GTPase EFL1 and the SBDS protein. These proteins participate in the synthesis of ribosomes by promoting the release of eukaryotic initiation factor 6 from the surface of the 60S ribosomal subunit 8. The SBDS protein is mutated in a disease known as the Shwachman-Diamond Syndrome 9 and acts as a guanine nucleotide exchange factor for EFL1 decreasing its affinity for guanosine diphosphate 10,11. Disease mutations in SBDS abolish the interaction with EFL1 and thus prevent its activation.
Fluorescence anisotropy is commonly used in biological applications to study protein-peptide or protein-nucleic acid interactions. It is based on the principle that a fluorophore excited with polarized light results in a partially polarized emission. Fluorescence anisotropy is defined by Equation 1:
<img alt="Equation1" src="/files/ftp_upload/54640/54640eq1.jpg" />
where IVV and IVH are the fluorescence intensities of the vertically (VV) and horizontally (VH) polarized emission when the sample is excited with vertically polarized light 12. Fluorescence anisotropy is sensitive to factors that affect the rate of the rotational diffusion of the fluorophore and thus depends on the temperature, the viscosity of the solution and the apparent molecular size of the fluorophore. The apparent size of a protein containing a fluorophore increases when it interacts with another protein and such change can then be evaluated as a change in anisotropy. More specifically, a fluorophore that rotates slowly in solution relative to its fluorescent lifetime will have a large IVV value and small IVH value and therefore will exhibit a relatively large anisotropy. For fluorophores that tumble rapidly relative to their fluorescent lifetime, IVV and IVH will be similar and their anisotropy value will be small 12 (Figure 1). In addition, for a good anisotropy signal to noise measurement, it is necessary to have a fluorophore with a fluorescence lifetime similar to the rotational correlation time of the molecule of interest. Otherwise, it is not possible to accurately record the difference in anisotropy between the free protein and that in the complex. For example, the anisotropy of a fluorescent probe with a lifetime close to 4 nsec such as fluorescein or rhodamine attached to a low molecular weight compound of 100 Da is 0.05. Binding to a molecule of 160 kDa will increase its anisotropy value to 0.29; a difference that can be accurately measured. In contrast, the same fluorescent probe involved in a binding reaction whose increase in molecular size varies from 65 to 1,000 kDa will only result in an anisotropy change of 0.28 to 0.3, which is too small to be accurately measured. In this scenario, a probe with a lifetime of 400 nsec would be more suitable 12.
<img alt="Figure 1" src="/files/ftp_upload/54640/54640fig1.jpg" /> 
Figure 1. Schematic representation of the equipment used to measure fluorescence anisotropy and the procedure. Schematic representation of the equipment used to perform a protein-protein interaction experiment measuring fluorescence anisotropy. Fluorophores that tumble fast display small anisotropy that increases upon binding to an interaction partner. <a href="http://cloudflare.jove.com/files/ftp_upload/54640/54640fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Fluorescence applications require the presence of a fluorophore in any of the molecules studied. To study protein-protein interactions there are three type of fluorophores: 1) the tryptophan residues present in the proteins, 2) chemically attached fluorophores and 3) fluorescent fusion partners such as green fluorescent protein (GFP) and its derivatives. Most proteins have tryptophan residues on its structure, thus the easiest way to measure an interaction is by monitoring the changes in the corresponding fluorescence spectra or by monitoring changes in the fluorescence intensity of the tryptophan residues. However, tryptophan residues may be present in both proteins complicating the analysis. On the other hand, for a fluorophore to change its fluorescent properties due to an interaction it needs to be located on or near the binding site and it could interfere with the interaction itself. This needs special attention when using bulky fluorophores such as GFP. If none of these fluorophores can be used for binding studies it is necessary, then, to introduce extrinsic fluorophores to the one of the proteins involved. Many chemically synthesized fluorophores exist and can be covalently attached to proteins through their reactive groups such as the amine groups (side chain of lysines or N-terminus) and the thiol groups in cysteine. Fluorophore derivatives with isothiocyanate and succinimidyl esters react with amide groups while iodoacetamide and maleimide are thiol-reactive groups 13. The most common dyes used in fluorescence applications are derivatives of the fluorescein and the rhodamine green dyes, coumarins, BODIPY fluorophores and Alexa Fluor dyes. A detailed list of commercially available fluorophores and their use can be found in references 14,15. For successful labeling, the reactive group must be exposed on the surface of the protein, but due to the large number of reactive functional groups typically present in polypeptides it is very hard to get site-specific modification. The protein of interest in this study, SBDS, contains 5 free cysteines and 33 lysines that may result in multiple site labeling. Non-uniform labeling may affect the binding and will complicate data analysis as different fluorophore molecules may elicit different fluorescent intensity signals upon binding. To overcome this problem, we used the FlAsH fluorophore, 4&#39;,5&#39;-bis(1,3,2 dithioarsolan-2-yl) fluorescein to site-direct label the SBDS protein. This is an arsenoxide dye with a high affinity for four spaced cysteines in a motif know as FlAsH-tag consisting of the sequence CCXXCC where X is any amino acid other than cysteine 16,17. This tetracysteine motif is added to the N- or C-terminus of the protein by genetic engineering together with an appropriate linker to prevent the disruption of the overall fold of the protein. The pair consisting of FlAsH dye and FlAsH-tag was originally designed to site-specific label proteins in living cells 17 but it can also be used to label purified proteins in vitro as it is exemplified here. Additionally, enzymatic strategies have also been developed to enable site-specific functionalization of proteins 18.
In this manuscript we describe the usefulness of fluorescence anisotropy as a tool to study protein-protein interactions. Binding can be assessed by simple inspection of the binding curve shape while quantitative information can be obtained from the fit of the experimental data.

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			<td style="width:119px;">CYN0410</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Yeast extract</td>
			<td style="width:112px;">Formedium</td>
			<td style="width:119px;">YEM03</td>
			<td style="width:225px;">Micro Granulated</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:219px;">L-Tyroisne</td>
			<td style="width:112px;">Formedium</td>
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			<td style="width:225px;"></td>
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		<tr height="21">
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			<td style="width:112px;">Formedium</td>
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			<td style="width:225px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Casamino acids</td>
			<td style="width:112px;">Formedium</td>
			<td style="width:119px;">CAS03</td>
			<td style="width:225px;"></td>
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		<tr height="42">
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			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">IPTG</td>
			<td style="width:112px;">Formedium</td>
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			<td style="width:225px;"></td>
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		<tr height="21">
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			<td style="width:112px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:225px;">Comments/Description</td>
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			<td style="width:119px;">GSWP04700</td>
			<td style="width:225px;">Membrane Filter, mixed cellulose esters, Hydrophilic, 0.22&#160;&#181;m, 47&#160;mm, white, plain</td>
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			<td height="42" style="height:42px;width:219px;">Ni2+ affinity column</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Strong Sulfopropyl cation exchanger column</td>
			<td style="width:112px;">GE Healthcare Life Science</td>
			<td style="width:119px;">17-5157-01</td>
			<td style="width:225px;">HiTrap SP Sepharose FF 5 ml</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Size Exclusion column</td>
			<td style="width:112px;">GE Healthcare Life Science</td>
			<td style="width:119px;">28989335</td>
			<td style="width:225px;">HiLoad 16/600 Superdex 200 PG</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Fluorescence cell</td>
			<td style="width:112px;">Hellma Analytics</td>
			<td style="width:119px;">111-057-40</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Name of Equipment</td>
			<td style="width:112px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:225px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Spectrophotometer</td>
			<td style="width:112px;">Agilent Technologies</td>
			<td style="width:119px;">G6860AA</td>
			<td style="width:225px;">Cary 60 UV-Vis</td>
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		<tr height="63">
			<td height="63" style="height:63px;width:219px;">Shaker</td>
			<td style="width:112px;">ThermoFischer Scientific</td>
			<td style="width:119px;">SHKA4000-7</td>
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			<td height="42" style="height:42px;width:219px;">Centrifuge</td>
			<td style="width:112px;">ThermoFischer Scientific</td>
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			<td height="63" style="height:63px;width:219px;">FPLC</td>
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			<td height="42" style="height:42px;width:219px;">Spectrofluorometer</td>
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			<td style="width:119px;">No applicable</td>
			<td style="width:225px;">Olis DM 45 with Polarization Toolbox</td>
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</table>

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.

To perform any anisotropy experiment it is important to rule out large changes in the fluorescence intensity of the fluorophore since the observed anisotropy of a mixture of species is represented by Equation 5:
<img alt="Equation5" src="/files/ftp_upload/54640/54640eq5.jpg" />
where Fi represents the fractional fluorescence of each component and <e...

Watch this Scientific Journal Video about Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions at JoVE.com

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

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 ...

The overall goal of this experiment is to evaluate and quantify the interaction between two proteins of interest using Fluorescence Anisotropy. These measures can help answer key questions. The protein biochemistry and protein biophysics field, such as protein whose interactions are important for cellular function.The main advantage of this technique is that we can obtain quantitative and qualitative information on the protein-protein interaction. To prepare purified SBDS-FlAsH protein, first prepare one liter of LB liquid medium supplemented with 100 micrograms per milliliter ampicillin. And in it, culture transformed bacteria at 37 degrees Celsius until the optical density at 600 nanometers reaches 0.5 to 0.7.Induce SBDS-FlAsH protein expression by adding 0.5 millimolar IPTG to the culture and continue the incubation for five more hours. Next, collect the bacterial suspension by centrifugation at 3800 times G for ten minutes at four degrees Celsius. Remove the supernatant.Re-suspend the cells in 35 milliliters of SBDS lysis butter supplemented with one millimolar PMSF. Lyse by sonication for a total time of four minutes using cycles of ten seconds on and 30 seconds off at four degrees Celsius. Then, centrifuge the sample at 9, 000 times G for 50 minutes at four degrees Celsius.Keep the supernatant and discard the palette to remove cellular debris. After equilibrating the nickel affinity column with three column volumes of SBDS lysis buffer, introduce the whole clarified supernatant onto the column. Remove the unbound protein by washing with three column volumes of SBDS lysis buffer.Following the column wash, elute the bound protein with three column volumes of SBDS elution buffer. To further purify the protein, equilibrate the sulfopropyl cation exchanger column with three column volumes of Low salt S column buffer. Dilute the protein six-fold with 50 millimolar phosphate buffer PH 6.5 and introduce it to the column.Wash the unbound material with three column volumes of Low salt S column buffer. Then, elute the protein in one step by adding 50 millimolar phosphate buffer PH 6.5, one molar sodium chloride onto the column. Dilute the eluate three-fold with 50 millimolar phosphate buffer PH 6.5.Then, concentrate the protein with ultra filtration devices by centrifugation at 3800 times G for fifteen minutes. The quality of the proteins used in this type of experiment are very potent. Interpretation of the data gets complicated if the protein samples used are not of high purity.Verify the purity of the SBDS-FlAsH protein by SDS-PAGE analysis and Coomassie staining. Flash-freeze the protein in liquid nitrogen and store it at minus 80 degrees Celsius until further use. To prepare purified EFL1 protein, first culture transformed yeast at 30 degrees Celsius in one liter of synthetic drop-out medium without uracil, supplemented with 0.5 percent glucose until the optical density at 600 nanometers reaches 1.8.Induce protein expression by adding 2.8 percent galactose to the culture and continue the incubation for 18 hours at 30 degrees Celsius. Collect the yeast suspension by centrifugation at 3800 times G for ten minutes at four degrees Celsius. Remove the supernatant following centrifugation.Re-suspend the cells in 50 milliliters of EFL1 lysis buffer supplemented with one millimolar PMSF and one millimolar benzamidine. Disrupt the cells by friction on a bead-beater using glass beads for a total time of six minutes using cycles of two minutes on and fifteen minutes off at four degrees Celsius. Then, centrifuge the sample at 9, 000 times G for 50 minutes at four degrees Celsius.Keep the supernatant and discard the palate to remove cellular debris. Next, equilibrate the nickel affinity column with three column volumes of EFL1 lysis buffer. Introduce all the clarified supernatant onto the equilibrated column.After removing unbound protein by washing the column with three column volumes of EFL1 lysis buffer, elute the bound protein with three column volumes of EFL1 elution buffer. To further purify the EFL1 protein, equilibrate the size exclusion column with 1.5 column volumes of Anisotropy buffer. Concentrate the EFL1 protein eluded from the nickel affinity column to one milliliter with ultra filtration devices by centrifugation at 3800 times G.Then, introduce the concentrated sample onto the size exclusion column.Collect the eluded protein and concentrate by ultra filtration to a final concentration of approximately 30 micromolar. Verify the purity of the EFL1 protein by SDS-PAGE analysis and Coomassie staining. Flash-freeze the protein in liquid nitrogen and store at minus 80 degrees Celsius until further use.Mix three nanomoles of the SBDS-FlAsH protein with three nanomoles of the lumio green dye in a five micrometer volume of anisotropy buffer. Let the reaction proceed for eight hours at four degrees Celsius. After eight hours, dialyze the sample against the anisotropy buffer overnight to remove the free dye.Measure the absorbents at 280 nanometers and 508 nanometers in a spectrophotometer using a Quartz Cuvette. Then, use the Lambert-Beer Law to quantify the percent of labeled protein as described in the text protocol. Before measuring the anisotrophy value, each filtration step from the protein legion should be done carefully, ensuring that the entire sample is dispensed into the solution and becomes homogeneous.In a Fluorescence Cuvette, place 200 microliters of 30 nanomolar SBDS-FlAsH in anisotrophy buffer and titrate two microliters of 30 micromolar EFL1. Mix thoroughly, and let the reaction stand for three minutes before measuring the anisotrophy and fluorescence value. Repeat this process until a total volume of 40 microliters of EFL1 has been added.As a final step, fit the data to a presumed binding model as described in the text protocol. To perform any anisotrophy experiment it is important to rule out large changes in the fluorescence intensity. As shown here, the fluorescence of SBDS-FlAsH does not noticeably change upon binding to EFL1.Titration of EFL1 into a Quartz Cuvette containing SBDS-FlAsH results in an increase of the initial observed anisotrophy. Several additions of EFL1 describe the corresponding binding curve that can be fit using non-linear least squares regression to a presumed binding model. In this case, a one binding site model does not appropriately describe the experimental data.Instead, a two non-identical binding sites model does appropriately describe the experimental data. Once mastered, the fluorescence anisotrophy binding experiment can be done in three hours if it is performed properly. While attempting this procedure, it is important to have purified protein in large quantities.In parallel with this procedure, other methods like this used to have with fragment-based complementation assay, or fluorescence anisotrophy with different domains of the studied protein can be performed in order to support the appropriate binding model. After watching this video, you should have a good understanding of how to perform a binding experiment followed by fluorescence anisotrophy.

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