Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

Summary

Protein interactions are at the heart of a cell'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).

Abstract

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=(I_VV-I_VH)/(I_VV+2I_VH), where I_VV and I_VH 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',5'-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.

Introduction

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:

where I_VV and I_VH 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 I_VV value and small I_VH value and therefore will exhibit a relatively large anisotropy. For fluorophores that tumble rapidly relative to their fluorescent lifetime, I_VV and I_VH 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.

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. Please click here to view a larger version of this figure.

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',5'-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.

Protocol

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.

1. SBDS-FlAsH protein expression
   1. Transform competent E. coli C41 cells with the plasmid pRSET-HisSBDS-FlAsH using a standard heat shock protocol ¹⁹. Plate the cells in solid Luria-Bertani (LB) media supplemented with 100 µg/ml ampicillin. LB solid media composition consists of 10 g NaCl, 5 g yeast extract, 10 g tryptone and 20 g agar for 1 L volume.
   2. Culture transformed bacteria at 37 °C until absorbance at 600 nm (A₆₀₀) reaches 0.5-0.7 in 1 L of LB liquid media supplemented with 100 µg/ml ampicillin.
   3. Induce protein expression by adding 0.5 mM isopropyl β-D-1-thiogalactopyranoside to the culture and continue the incubation for further 5 hr.
   4. Collect the bacterial suspension by centrifugation at 3,800 x g for 10 min at 4 °C. Remove the supernatant. At this point, either store the cell pellet at -20 °C or use immediately for protein purification.
2. SBDS-FlAsH protein purification
   NOTE: All chromatographic steps are performed using a fast protein liquid chromatography (FPLC) system or a peristaltic pump. Ni²⁺-affinity chromatography uses a 5 ml fast flow column. Anionic exchange chromatography uses a 5 ml strong sulfopropyl cation exchanger column. A flow rate of 3 ml/min was used for all the chromatographic steps.
   1. Resuspend the cells in 35 ml of SBDS Lysis buffer (50 mM phosphate buffer pH 7.5, 300 mM NaCl, 20 mM imidazole) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) and lyse by sonication for a total time of 4 min using cycles of 10 sec ON and 30 sec OFF, at 4 °C.
   2. Centrifuge the sample at 9,000 x g for 50 min at 4 °C.
   3. Keep the supernatant and discard the pellet to remove cellular debris.
   4. Equilibrate the Ni²⁺ affinity column with 3 column volumes (CV) of SBDS Lysis buffer and introduce the whole clarified supernatant onto the column.
   5. Remove unbound protein by washing with 3 CV of SBDS Lysis buffer and elute with 3 CV of SBDS Elution buffer (50 mM phosphate buffer pH 7.5, 300 mM NaCl, 250 mM imidazole).
   6. Dilute the eluted protein 6-fold with 50 mM Phosphate buffer pH 6.5 and remove possible aggregates by filtration through a 0.22 µm cellulose membrane.
   7. Equilibrate the sulfopropyl cation exchanger column with 3 CV of Low salt S column buffer (50 mM phosphate buffer pH 6.5, 50 mM NaCl) and introduce the protein sample from the previous step.
   8. Wash unbound material with 3 CV of Low salt S column buffer and elute the protein in one step with 50 mM phosphate buffer pH 6.5, 1 M NaCl.
   9. Dilute the eluted protein 3.3-fold with 50 mM Phosphate buffer pH 6.5. Concentrate the protein with ultrafiltration devices by centrifugation at 3,800 x g for 15 min. Flash freeze the protein in liquid nitrogen and store it at -80 °C until further use.
   10. Verify the purity of the protein by SDS-PAGE analysis and Coomassie staining ²⁰.

2. EFL1 Protein Expression and Purification

NOTE: EFL1 was expressed under the regulation of the Gal 1/10 divergent promoter ²¹ and the Saccharomyces cerevisiae MATA 3'UTR in the vector pRS426. The recombinant protein encodes the human EFL1 isoform 1 fused to a Tobacco Etch Virus protease (TEV) recognition site and a hexahistidine tag at the C-terminus.

1. EFL1 protein expression
   1. Transform S. cerevisiae BCY123 cells with the plasmid pRS426-EFL1TevHis using a standard Lithium acetate protocol ²². Plate all the transformed cells in synthetic drop out media without uracil (SD-URA) supplemented with 2% (w/v) glucose. Composition of the SD-URA media consists of 8 g yeast nitrogen base without amino acids, 11 g casamino acids, 55 mg adenine sulfate, 55 mg tyrosine, 60 mg leucine and 60 mg tryptophan for 1 L volume.
   2. Culture transformed yeast at 30 °C until A₆₀₀ reaches 1.8 in 1 L of SD-URA media supplemented with 0.5% (w/v) glucose.
   3. Induce protein expression by adding 2.8% (w/v) galactose to the culture and continue the incubation for further 18 hr at 30 °C.
   4. Collect the yeast suspension by centrifugation at 3,800 x g for 10 min at 4 °C. Remove the supernatant. At this point, either store the cell pellet at -20 °C or use immediately for protein purification.
2. EFL1 protein purification
   NOTE: All chromatographic steps are performed using a FPLC system or a peristaltic pump. Ni²⁺-affinity chromatography uses a 5 ml fast flow column at a flow rate of 3 ml/min. Size exclusion chromatography uses a 125 ml column pre-packed with Superdex 200 resin at a flow rate of 1 ml/min.
   1. Resuspend the cells in 50 ml of EFL1 Lysis buffer (50 mM Tris-HCl pH 8, 300 mM NaCl, 20 mM imidazole, 5 mM MgCl₂, 10% glycerol) supplemented with 1 mM PMSF and 1 mM benzamidine and disrupt the cells by friction on a bead beater using glass beads (Ø = 0.5 mm) for a total time of 6 min using cycles of 2 min ON and 15 min OFF, at 4 °C.
   2. Centrifuge the sample at 9,000 x g for 50 min at 4 °C.
   3. Keep the supernatant and discard the pellet to remove cellular debris.
   4. Equilibrate the Ni²⁺ affinity column with 3 CV of EFL1 Lysis buffer and introduce all the clarified supernatant onto the column.
   5. Remove unbound protein by washing with 3 CV of EFL1 Lysis buffer and elute with 3 CV of EFL1 Elution buffer (50 mM Tris-HCl pH 8, 300 mM NaCl, 250 mM imidazole, 5 mM MgCl₂, 10% glycerol).
   6. Equilibrate the size exclusion column with 1.5 CV of Anisotropy buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 5 mM MgCl₂, 10% glycerol, 5 mM β-mercaptoethanol).
   7. Concentrate to 1 ml the EFL1 protein eluted from the Ni²⁺ affinity column with ultrafiltration devices by centrifugation at 3,800 x g to the desired volume. Introduce the sample on the size exclusion column.
   8. Collect the eluted protein and concentrate by ultrafiltration to a final concentration of approximately 30 µM. Flash freeze the protein in liquid nitrogen and store at -80 °C until further use. Verify the purity of the protein by SDS-PAGE analysis and Coomassie staining ²⁰.

3. Labeling of SBDS-FlAsH with the FlAsH Fluorescent Dye 4',5'-Bis(1,3,2 dithioarsolan-2-yl) Fluorescein

1. Mix 3 nmol of the SBDS-FlAsH protein with 3 nmol of the 4',5'-bis(1,3,2 dithioarsolan-2-yl) fluorescein dye in 5 µl volume of Anisotropy buffer.
2. Let the reaction proceed for 8 hr at 4 °C. Dialyze the sample against Anisotropy buffer over night to remove the free dye.
3. Use the Lambert-Beer law to quantify the % of labeled protein. Measure the absorbance at 280 nm and 508 nm in a spectrophotometer using a quartz cuvette of appropriate volume. NOTE: Consider the following molar absorption coefficients (M^-1 cm^-1):
   
4. Calculate the concentration of labeled SBDS-FlAsH protein using Equation 2.
   
5. Calculate the concentration of total SBDS protein using Equation 3 by substituting the calculated C_SBDS-FlAsH from previous step.
   
6. Calculate the percentage of labeled protein using Equation 4.
   

4. Fluorescence Anisotropy Experiments

NOTE: Anisotropy experiments were done in a spectrofluorometer equipped with a polarization toolbox and data collection was performed using the anisotropy program provided in the software of the equipment. The excitation wavelength was set at 494 nm with a spectral bandwidth of 8 nm and the emission was recorded using a band-pass filter of 530±25 nm. Measurements were done at 25 °C in a 200 µl cuvette with a 5 mm path length ¹⁰.

1. In a fluorescence cuvette, place 200 µl of 30 nM SBDS-FlAsH in anisotropy buffer and titrate 2 µl of 30 µM EFL1. Mix thoroughly and let the reaction stand for 3 min before measuring the anisotropy value.
2. Repeat step 4.1 until a total volume of 40 µl of EFL1 has been added.

5. Data Analysis

1. Fit the data to the appropriate binding model using a nonlinear least squares regression algorithm. Equations for the most common binding models are presented in Table 1.
2. Evaluate the best model that describes the interaction between the proteins by inspecting the residuals of the fit ²³. Support the chosen model with additional experiments.

Table 1. Common protein-protein interaction binding models and the mathematical equations that describe them. Please click here to view a larger version of this table.

Results

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:

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.5 mm Glass beads	Biospec Products	11079105
Tris Base	Formedium	TRIS01	Ultra pure
Glycerol	Sigma-Aldrich	G5516
dye 4’,5’-bis(1,3,2 dithioarsolan-2-yl) fluorescein	ThermoFischer Scientific	LC6090	This kit contains the dye to label a FlAsH tag
Ampiciline	IBI Shelton Scientific, Inc	IB02040
D(+)-Glucose Anhydrous	Formedium	GLU03
D(+)-Galactose	Formedium	GAL03
L-Leucine	Formedium	DOC0157
L-Tryptofan	Formedium	DOC0189
Bezamidine hydrochloride	Sigma-Aldrich	B6506-5G
PMSF	Gold Biotechnology, Inc	P-470-25	Phenylmethylsulfonyl fluoride
NaCl	Formedium	NAC02	Sodium Chloride
Glycerol	Tecsiquim, S.A. de C.V.	GT1980-6
MgCl2	Merck Millipore Corporation	1725711000	Magnesium Chloride
Imidazole	Sigma-Aldrich	I2399-500G
2-Mercaptoethanol	Sigma-Aldrich	M6250-100ML
K2HPO4	Sigma-Aldrich	P3786-500G	Potassium phosphate dibasic
NaH2PO4	Sigma-Aldrich	S3139-500G	Sodium phosphate monobasic
Yeast nitrogen base without amino acids	Formedium	CYN0410
Yeast extract	Formedium	YEM03	Micro Granulated
L-Tyroisne	Formedium	DOC0193
Adenine sulphate	Formedium	DOC0230
Casamino acids	Formedium	CAS03
Tryptone	IBI Shelton Scientific, Inc	IB49182
IPTG	Formedium	IPTG025
Filtration units	Merck Millipore Corporation	UFC901096	Amicon Ultra-15, membrana PLGC Ultracel-PL, 10 kDa
Membrane Filter	Merck Millipore Corporation	GSWP04700	Membrane Filter, mixed cellulose esters, Hydrophilic, 0.22 µm, 47 mm, white, plain
Ni2+ affinity column	QIAGEN	30760	Cartridge pre-filled with 5 ml Ni-NTA Superflow
Strong Sulfopropyl cation exchanger column	GE Healthcare Life Science	17-5157-01	HiTrap SP Sepharose FF 5 ml
Size Exclusion column	GE Healthcare Life Science	28989335	HiLoad 16/600 Superdex 200 PG
Fluorescence cell	Hellma Analytics	111-057-40
Spectrophotometer	Agilent Technologies	G6860AA	Cary 60 UV-Vis
Shaker	ThermoFischer Scientific	SHKA4000-7	MaxQ 4000 Benchtop temperature range Ambient-15° to 60°C
Centrifuge	ThermoFischer Scientific	75004271	Heraeus Megafuge 16R
FPLC	Pharmacia Biotech	Discontinued	FPLC system conductivity UV-MM II monitor P500 pump fraction
Spectrofluorometer	Olis	No applicable	Olis DM 45 with Polarization Toolbox