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).
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',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.
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 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.
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.
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…
The authors have nothing to disclose.
Authors acknowledge the financial support from CONACyT project numbers 167359 and 177138, and from DGAPA-UNAM project number IN201615.
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 | |
Name of Material | Company | Catalog Number | Comments/Description |
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 | |
Name of Equipment | Company | Catalog Number | Comments/Description |
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 |