Fluorescence Anisotropy-Based Detection of Protein-Protein Interactions

Overview

In this video, we describe the fluorescence anisotropy technique to study the interactions between the fluorophore-tagged Shwachman-diamond syndrome (SBDS) protein and the elongation factor-like 1 GTPase (EFL1). On incubating SBDS proteins with gradually increasing concentrations of EFL1, a steady increase in anisotropy is observed, indicating a successful interaction between the two proteins.

Protocol

1. 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 (the FlAsH-tag corresponds to the tetracysteine motif Cys-Cys-Pro-Gly-Cys-Cys genetically engineered in the C-terminus of the recombinant SBDS protein) with 3 nmol of the 4',5'-bis(1,3,2 dithioarsolan-2-yl) fluorescein dye in 5 µl volume of Anisotropy buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 5 mM MgCl₂, 10% glycerol, 5 mM β-mercaptoethanol).
2. Let the reaction proceed for 8 hr at 4 °C. Dialyze the sample against Anisotropy buffer overnight 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):
     Equation 1
4. Calculate the concentration of labeled SBDS-FlAsH protein using Equation 2.
     Equation 2
5. Calculate the concentration of total SBDS protein using Equation 3 by substituting the calculated C_SBDS-FlAsH from the previous step.
     Equation 3
6. Calculate the percentage of labeled protein using Equation 4.
     Equation 4

2. 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.

1. In a fluorescence cuvette, place 200 µl of 30 nM SBDS-FlAsH in an 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 2.1 until a total volume of 40 µl of EFL1 has been added.

3. 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. Support the chosen model with additional experiments.

Table 1. Common protein-protein interaction binding models and the mathematical equations that describe them.

Materials

Name	Company	Catalog Number	Comments
Tris Base	Formedium	TRIS01	Ultra pure
dye 4’,5’-bis(1,3,2 dithioarsolan-2- yl) fluorescein	ThermoFischer Scientific	LC6090	This kit contains the dye to label a FlAsH tag
Fluorescence cell	Hellma Analytics	111-057-40
Spectrophotometer	Agilent Technologies	G6860AA	Cary 60 UV-Vis
Spectrofluorometer	Olis	No applicable	Olis DM 45 with Polarization Toolbox
2-Mercaptoethanol	Sigma-Aldrich	M6250-100ML
NaCl	Formedium	NAC02	Sodium Chloride
MgCl2	Merck Millipore Corporation	1725711000	Magnesium Chloride
Glycerol	Sigma-Aldrich	G5516
Glycerol	Tecsiquim, S.A. de C.V.	GT1980-6