Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

Robert P. Sparks; William Lawless; Andres  S. Arango; Emad Tajkhorshid; Rutilio  A. Fratti

doi:10.3791/60607

Utilisation de la thermophorèse à micro-échelle pour mesurer les interactions protéine-lipide

JoVE Journal
Biochemistry

A subscription to JoVE is required to view this content. Sign in or start your free trial.

JoVE Journal Biochemistry

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

Utilisation de la thermophorèse à micro-échelle pour mesurer les interactions protéine-lipide

Full Text

7,994 Views

04:45 min

February 10, 2022

DOI: 10.3791/60607-v

Robert P. Sparks*¹, William Lawless*², Andres S. Arango*^1,3,4, Emad Tajkhorshid^1,3,4, Rutilio A. Fratti^1,3

¹Department of Biochemistry,University of Illinois at Urbana-Champaign, ²Department of Chemistry,University of South Florida, ³Center for Biophysics and Quantitative Biology,University of Illinois at Urbana-Champaign, ⁴Beckman Institute for Advanced Science and Technology,University of Illinois at Urbana-Champaign

Please note that some of the translations on this page are AI generated. Click here for the English version.

Overview

Labeled microscale thermophoresis is an efficient method for obtaining disassociation constants (KDs) for various biochemical interactions. This protocol focuses on the affinity measurement of Vam7, a soluble SNARE protein in yeast, with phosphatidic acid, highlighting its role in membrane interactions.

Key Study Components

Area of Science

Biochemistry
Biophysics
Pharmaceutical Development

Background

Microscale thermophoresis is a technique for measuring molecular interactions.
Labeled thermophoresis provides more diverse interaction measurements than label-free methods.
Understanding protein-lipid interactions is crucial for cellular processes.
Vam7 is the only soluble SNARE protein in yeast, essential for membrane fusion.

Purpose of Study

To provide a protocol for measuring KDs using labeled microscale thermophoresis.
To demonstrate the interaction between Vam7 and phosphatidic acid.
To facilitate early-stage pharmaceutical development by providing rapid affinity measurements.

Methods Used

Preparation of labeled Vam7 protein.
Use of microscale thermophoresis device for interaction measurement.
Analysis of binding affinities with phosphatidic acid.
Software control for data acquisition and analysis.

Main Results

Efficient determination of KDs for Vam7 and phosphatidic acid.
High reproducibility and low material costs associated with the method.
Demonstrated suitability for early-stage drug development.
Protocol allows for rapid acquisition of binding data.

Conclusions

Labeled microscale thermophoresis is a valuable tool for studying protein interactions.
The method provides quick and cost-effective results.
Understanding Vam7 interactions can inform drug discovery efforts.

Frequently Asked Questions

What is microscale thermophoresis?

Microscale thermophoresis is a technique used to measure molecular interactions by observing the movement of molecules in a temperature gradient.

How does labeled thermophoresis differ from label-free methods?

Labeled thermophoresis allows for a wider range of interaction measurements compared to label-free methods, which are limited in their capabilities.

What is the significance of Vam7 in yeast?

Vam7 is crucial for membrane fusion processes in yeast, making it an important protein for studying cellular interactions.

What are disassociation constants (KDs)?

KDs are measures of the affinity between two molecules, indicating how readily they associate and dissociate from each other.

How can this method be applied in pharmaceutical development?

This method allows for rapid and cost-effective determination of binding affinities, aiding in the identification of potential lead compounds in drug development.

La thermophorèse à micro-échelle permet d’obtenir rapidement des constantes de liaison à faible coût matériel. La thermophorèse micro-échelle étiquetée ou sans étiquette est disponible dans le commerce; cependant, la thermophorèse sans étiquette n’est pas capable de la diversité des mesures d’interaction qui peuvent être effectuées à l’aide d’étiquettes fluorescentes. Nous fournissons un protocole pour les mesures de thermophorèse marquées.

La thermophorèse à micro-échelle marquée permet d’obtenir efficacement des constantes de dissociation, ou KD, de diverses interactions biochimiques. Ce protocole et la vidéo accompagnée donnent l’affinité approximative de Vam7, la seule protéine SNARE soluble dans la levure, à l’acide phosphatidique. C’est ainsi que Vam7 s’associe à deux membranes avant d’interagir avec d’autres protéines.

Cette méthode permet d’obtenir rapidement des KD pour diverses interactions biochimiques à faible coût, avec une reproductibilité élevée et un coût protéique minimal. Cette technique convient au développement pharmaceutique de première étape, permettant d’obtenir facilement des KD pour déterminer les composés principaux potentiels. Avant de commencer une analyse, allumez l’interrupteur d’alimentation à l’arrière du périphérique MST et ouvrez le logiciel de contrôle.

View the full transcript and gain access to thousands of scientific videos

Explore More Videos

Biochimie Numéro 180 Phosphoinositide Phosphatidylinositol 3-phosphate domaine FYVE Vacuole Lysosome Endosome