Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

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).

Transcript

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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Journal (Biochemistry)

Journal (Biochemistry)

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

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