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. Howeve....

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 mo....

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