Microscale Thermophoresis to Study Protein-Lipid Interactions in Solution

Overview

This video demonstrates microscale thermophoresis for studying the protein-lipid interaction. The assay detects the interaction between molecules by quantifying the thermophoretic movement of fluorescent-labeled proteins in response to a temperature gradient. The fluorescent molecule is mixed with different concentrations of the non-fluorescent lipid molecules, and the mixture of molecules in the solution is loaded into capillaries. A temperature gradient is applied to the samples in the capillaries, and the movement of the fluorescent molecule in the temperature gradient is detected and recorded.

Protocol

1. Preparation of materials

1. Prepare phosphate-buffered saline (PBS): 137 mM NaCl, 2.5 mM KCl, 10 mM NaH₂PO₄, and 2 mM KH₂PO_4, pH 7.4.
2. Prepare NTA-Atto 647 N dye. Dilute stock NTA-Atto 647 N dye to 100 nM from a 100% DMSO solution into PBS without Tween.
3. Express Vam7-His8 – Protein as a fusion protein in E. coli and purify it using Ni-NTA and size exclusion chromatography.
4. Titrate the analyte in PBS buffer. If the analyte is in a different buffer, labeled MST is not particularly sensitive to minor buffer differences from molecules such as DMSO, unless the contents of the analyte buffer interact with the labeled protein.

2. Preparation of the MST device

1. Turn on the power switch on the back of the device.
2. Open the control software and ensure that the laptop is on and in connected status on the computer attached to the device.
3. Enter fluorescence and MST settings for this experiment. Set MST Before to 3 s, MST to 30 s, and Fluorescence recovery (Fluo.) after 1 s. Before measures initial fluorescence and does not require long times; MST is the actual amount of time for equilibrium to be reached after heat induction.
4. Table of Capillaries: 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 and use the autofill titration ratio. For example, enter 50 nM for the target concentration of Vam7-His8, Vam7-His8 for the target name, (1,2-dioctanoyl phosphatidic acid) Di-C8 PA for the ligand name, and the highest concentration of the ligand selecting 1:1 and dragging down to autofill slots 2-16.
5. Run a Cap Scan to select the appropriate LED (20% LED (preset)) based on the signal of the target protein and adjust between 200 and 2000 fluorescence units for labeled MST. Cap Scans should show uniform rounded bell-shaped peaks.
6. Select a range of MST powers and enter values for each to test for the most robust binding fit and hit Start Cap Scan + Measurement, scanning different values to determine the best operating conditions for the given interaction being tested.
7. Determine the MST power with the best fit using the analysis software and the preset for thermophoresis with Tjump. Analyze fits according to most fluorescence separation between lowest and highest concentration as compared to the MST power with the best fit and select the MST power for replicate trials.
8. Determine whether photobleaching has occurred between the first and second run by going to the analysis software and switching the analysis to expert mode. Next, select fluorescence instead of thermophoresis for analysis. Select expert mode and then photobleaching.

3. Preparation of samples for labeled MST

1. Bring the Vam7-His8 solution to a concentration of 200 nM in PBS without Tween.
2. Bring the NTA-Atto 647 dye to 100 nM in PBS without Tween.
3. Mix the Vam7-His8 and NTA-Atto 647 dye at a 1:1 volumetric ratio and allow it to sit at room temperature covered from light for 30 min. Determine the appropriate concentration of target protein as in section 2.5 (see Supplement on utilization of KD of Ni-NTA dye as demonstrated in the video).
4. Centrifuge mixture of NTA-Atto 647 N dye and protein for 10 min in a dark room using a tabletop centrifuge at approximately 8,161 x g.
5. Store the mixture at 4 °C after the experiment or on ice during the experiment for reuse within a few hours if needed in order to keep protein from denaturing.
6. Use the NT concentration finder to determine the concentration range needed for titration.
7. Bring Di-C8 PA to the appropriate maximum concentration in water.
8. Titrate the analyte using a 1:1 serial dilution in PBS buffer for 16 concentrations based on the previous step. Unlike SPR, thermophoresis is not as sensitive to buffer differences.

4. MST of samples

1. Turn on the device and the attached laptop.
2. Press the up arrow on the front of the machine and slide the capillary rack out.
3. Load capillaries in the rack with the highest concentration at position 1.
4. In the control software, select the Red Channel corresponding to NTA-Atto 647 N.
5. Enter concentration, position, and name information for each capillary in the Table of Capillaries.
6. Run a capillary scan by hitting Start Cap Scan at 20% LED (preset) and adjust accordingly between 200 and 2000 fluorescence units using either LED intensity settings or concentration of ligand (labeled protein).
7. Select a range of MST power.
8. Start Cap Scan + MST Measurement.
9. Analyze using the analysis software.

Materials

Name	Company	Catalog Number	Comments
Cy5 Maleimide Mono-Reactive Dye	GE Healthcare	PA23031	For protein labeling
GraphPad Prism	GraphPad software
Monolith NT.115 Capillaries (1000 count)	Nanotemper	MO-K022	Capillaries for MST
Monolith NT.115 machine	Nanotemper		University equipment
NTA-Atto 647 N	Sigma	2175	Label for His tags
Phosphatidylinositol 3-phosphate diC8 (PI(3)P diC8)	Echelon	P-3008	Lipid for binding experiments