Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

Summary

Microscale thermophoresis obtains binding constants quickly at low material cost. Either labeled or label free microscale thermophoresis is commercially available; however, label free thermophoresis is not capable of the diversity of interaction measurements that can be performed using fluorescent labels. We provide a protocol for labeled thermophoresis measurements.

Abstract

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane trafficking, signal transduction and cytoskeletal remodeling. Classic tools for measuring such interactions include surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). While powerful tools, these approaches have setbacks. ITC requires large amounts of purified protein as well as lipids, which can be costly and difficult to produce. Furthermore, ITC as well as SPR are very time consuming, which could add significantly to the cost of performing these experiments. One way to bypass these restrictions is to use the relatively new technique of microscale thermophoresis (MST). MST is fast and cost effective using small amounts of sample to obtain a saturation curve for a given binding event. There currently are two types of MST systems available. One type of MST requires labeling with a fluorophore in the blue or red spectrum. The second system relies on the intrinsic fluorescence of aromatic amino acids in the UV range. Both systems detect the movement of molecules in response to localized induction of heat from an infrared laser. Each approach has its advantages and disadvantages. Label-free MST can use untagged native proteins; however, many analytes, including pharmaceuticals, fluoresce in the UV range, which can interfere with determination of accurate K_D values. In comparison, labeled MST allows for a greater diversity of measurable pairwise interactions utilizing fluorescently labeled probes attached to ligands with measurable absorbances in the visible range as opposed to UV, limiting the potential for interfering signals from analytes.

Introduction

Microscale thermophoresis is a relatively new technique in determining disassociation constants (K_D) as well as inhibition constants (IC₅₀) between biochemically relevant ligands. The leading commercial retailer for MST (e.g., NanoTemper) offers two popular MST technologies: 1) Label free MST requiring a fluorescent tag, and 2) labeled thermophoresis using the inherent fluorescence of proteins dependent on the number of aromatic residues present in each protein¹. A disadvantage of label-free thermophoresis is that in most cases, it does not allow for the measurement of protein-protein interactions. However, it may be possible to engineer proteins without aromatic amino acids such as tryptophan for use in label free thermophoresis².

MST measures the movement of particles in response to the induction of microscopic temperature fields initiated by an infrared laser in currently available technologies¹. MST can be used to measure protein-protein interactions, protein-lipid interactions, protein-small molecule, competition experiments, and even interactions between small-molecules so long as one can produce enough signal separation. Additionally, MST allows for the measurement of membrane-protein based interactions embedded in either liposomes or nanodiscs. Labeled thermophoresis takes advantage of the use of fluorescently labeled tags allowing for chemically controllable separation of signal between ligand and analyte. K_D values can be obtained using thermophoresis for interactions involving protein binding at low nanomolar concentrations, which in most cases is a much lower concentration of protein than what is required for isothermal calorimetry (ITC)³. Additionally, MST does not have strict buffering requirements as required for surface plasmon resonance (SPR)⁴ and labeled thermophoresis can even be used to measure binding constants of proteins of interest from non-fully purified protein solutions⁵ with genetically inserted fluorescent tags⁶. A disadvantage of MST is that kinetic parameters cannot be obtained readily for MST as in SPR².

Thermophoresis measurements depend on the local temperature difference of a solution. This heat can be generated from an infrared laser. The MST device has a fluorescence detector coupled to an infrared (IR) beam and can pick up changes in fluorescence from local concentration changes of the fluorescent molecules at the point where the IR laser is targeted. The MST device utilizes an IR targeted laser coupled directly to a fluorescence detector focused at the same point in which the heat is generated in the solution. This allows for robust detection of changes in temperature corresponding to the depletion of molecules at the point of heat generated by the IR laser. Measured fluorescence generally decreases closer to the IR laser in response to temperature increases. The differences measured as a result can be due to multiple factors including charge, size, or solvation entropy. These differences are measured as changes in fluorescence in response to induction of heat or movement of molecules from hot to colder parts of the capillary.

When loading a capillary with a given solution, it is important to leave air at either end of the capillary and not load the capillary completely full. The commercial capillary holds about 10 μL of solution. One can achieve accurate measurements with 5 μL of solution so long as the solution is manipulated to the center of the capillary, there are no air bubbles (potentially degas prior to loading capillary), and one is careful loading the rack to not jostle the solution from the center of the capillary, where the infrared laser is targeted. If the laser does not come in contact fully with solution, the result will most likely be one of three unusable outputs for that concentration: 1) no or low fluorescence detection, 2) higher fluorescence detection (potentially with jagged peak), or 3) fluorescence detection within other values from given titration, but with a jagged and unrounded peak.

For labeled thermophoresis it is optimal to have a fluorescence signal above 200 and below 2000 fluorescence units⁷. The MST device uses a range of LED intensities from 0 to 100, which can be selected to achieve a signal above 200 or below 2000. Alternatively, one can use different concentrations of the labeled ligand to modify the fluorescence signal to an optimal level. It is important to run a cap scan with a given MST measurement as a reference when analyzing data, as a poor cap scan can often result in a point that may later be determined to be an outlier. Each run should take approximately 30 min if measuring a single MST power with a cap scan. The commercial devices allow changes in MST power. In older software versions this could be set from 0 to 100; and in later versions one can select low, medium, or high MST. To achieve robust traces, a researcher may need to try each of these and decide which MST setting results in the most robust data for a given interaction.

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 7.4¹.
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 using Ni-NTA and size exclusion chromatography⁸.
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 contents of analyte buffer interact with 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 on 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 autofill titration ratio. For example, enter 50 nM for the target concentration of Vam7-His8, Vam7-His8 for target name, (1,2-dioctanoyl phosphatidic acid) Di-C8 PA for ligand name, and the highest concentration 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 best operating conditions for given interaction being tested.
7. Determine the MST power with a 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 best fit and select the MST power for replicate trials.
8. Determine whether photobleaching has occurred between 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 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 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 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 concentration range needed for titration.
7. Bring Di-C8 PA to 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 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 according 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.

5. Analysis of MST data

NOTE: The analysis software provided by Nanotemper is proprietary and is performed using M.O. Affinity Analysis. There are different ways to measure binding affinities based on either fluorescence or thermophoresis. Newer versions of this software are preset to automatically evaluate data using thermophoresis and are preset to use Thermophoresis with Tjump taking advantage of both measurements. Alternatively, one can select either Tjump alone or Thermophoresis alone. Additionally, the analysis software allows estimated affinity measurements using initial fluorescence. These settings can be accessed in expert mode only.

1. Set the evaluation strategy in the analysis software to expert by clicking the box for the lightning bolt next to a data set in the Data Selection screen. The Analysis Software is preset to analyze to MST Analysis; however, a researcher can select Create New Analysis and select Initial Fluorescence Analysis in order to estimate binding affinities based on initial fluorescence. Expert mode is also available for initial fluorescence. In the analysis described below, thermophoresis and Tjump was used to determine the K_D of the presented of Vam7-His8 with its natural substrate, the lipid phosphatidic acid.

Results

This is a sample output using the affinity analysis. The labeled MST was used to determine the binding constant of the Vam7-His8 to the soluble dioctanoyl (DiC8) PA of one of its natural substrates⁹. Figure 1 presents the thermophoretic traces from one trial of a 1:1 titration of DiC8 PA starting at 500 μM against 50 nM of Vam7-His8. Initial fluorescence (time before infrared laser turned on), Tjump (time initially after infrared laser turned on), and thermo...

Discussion

The determination of Vam7-His8binding to DiC8-PA provided a robust fitted K_D for the given interaction, which is slightly lower affinity than the measured K_D of Vam7-His8 to PA liposomes (unpublished). This difference is most likely due to the lack of a membrane, which generally results in lower affinity for membrane specific lipid binding interactions and therefore demonstrates the role for the liposome membrane scaffold to this interaction

Materials

Name	Company	Catalog Number	Comments
Cy5 Maleimide Mono-Reactive Dye	GE Healthcare	PA23031	For protein labeleing
Graphpad Prsim	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