Labeled microscale thermophoresis obtains disassociation constants, or KDs, of diverse biochemical interactions efficiently. This protocol and the accompanied video give the approximate affinity for Vam7, the only soluble SNARE protein in yeast, to phosphatidic acid. This is the way that Vam7 associates with two membranes before interacting with other proteins.
This method obtains KDs rapidly for diverse biochemical interactions at low cost, with high reproducibility and with minimal protein cost. This technique is suitable for first-stage pharmaceutical development, allowing for readily obtainable KDs to determine potential lead compounds. Before beginning an analysis, turn on the power switch on the back of the MST device and open the control software.
Confirm that the computer is attached to the device and in connected status and set the MST Before to 3 seconds, the MST to 30 seconds, and the Fluorescence Recovery to after 1 second. For each capillary tube, enter the name of the target ligand, the name of the ligand analyte, the concentration of the target, and the highest titration concentration using the autofill titration ratio. Select a range of MST powers and enter values for each power to test for the most robust binding fit.
To prepare labeled MST samples, dilute a Vam7-octahistidine solution to a 200 nanomolar concentration and an NTA-Atto 647 dye to 100 nanomolar concentration, both in PBS. Mix the Vam7-octahistidine and the NTA-Atto 647 dye at a 1:1 volumetric ratio and allow the mixture to sit at room temperature protected from light for 30 minutes. At the end of the incubation, centrifuge the dye and protein mixture and store the solution at 4 degrees Celsius for up to a few hours before use.
Then, prepare the sample, take dyed ligand and expose the analyte. For microscale thermophoresis of the sample, open the device and slide the capillary rack out. Load the sample capillaries into the rack with the highest concentration at position one and select the Red channel in the control software.
Then, load the rack in the instrument. Then select a range of MST power and start the Cap Scan MST Measurement and evaluate the shift response. Thermophoretic traces from one trial of a 1:1 titration of DiC8 PA starting at 500 micromoles against 50 nanomolar NTA-Atto 647 labeled Vam7 domain are shown here.
The initial fluorescence, time jump, and thermophoresis can be observed, allowing KD to be calculated from any one or a combination of these measurements. A saturation curve can be plotted from these results, for example, for the thermophoresis with the time jump output, as illustrated in the graph. Generally, MST results are determined using log scale, taking into account the protein concentration to determine the binding affinity by selecting the KD model and inputting the protein concentration.
When performing this protocol, ensure that the solution is in the center of the capillary and that there's no air bubbles and that the capillary does not have dust or solution on the outside of the capillary. Isothermal titration calorimetry or surface plasmon resonance could be used to validate the experimental KD obtained. If there is potential cooperative binding, ITC would be the next logical method performed, given resources.
This technique has enabled traditional biology researchers to incorporate biophysical screening techniques into their research for determining pathway relationships. One example of this technique is cell lysase with the target protein expressed endogenously, where the GFP tag is a novel way to perform a traditional pull-down assay.
