Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

Summary

This video article details the experimental procedure for obtaining the Gibbs free energy of membrane protein folding by tryptophan fluorescence.

Abstract

Membrane protein folding is an emerging topic with both fundamental and health-related significance. The abundance of membrane proteins in cells underlies the need for comprehensive study of the folding of this ubiquitous family of proteins. Additionally, advances in our ability to characterize diseases associated with misfolded proteins have motivated significant experimental and theoretical efforts in the field of protein folding. Rapid progress in this important field is unfortunately hindered by the inherent challenges associated with membrane proteins and the complexity of the folding mechanism. Here, we outline an experimental procedure for measuring the thermodynamic property of the Gibbs free energy of unfolding in the absence of denaturant, ΔG°_H2O, for a representative integral membrane protein from E. coli. This protocol focuses on the application of fluorescence spectroscopy to determine equilibrium populations of folded and unfolded states as a function of denaturant concentration. Experimental considerations for the preparation of synthetic lipid vesicles as well as key steps in the data analysis procedure are highlighted. This technique is versatile and may be pursued with different types of denaturant, including temperature and pH, as well as in various folding environments of lipids and micelles. The current protocol is one that can be generalized to any membrane or soluble protein that meets the set of criteria discussed below.

Protocol

1. Preparation of ~50 nm Diameter Small Unilamellar Vesicles (SUVs) for Membrane Protein Folding

1. A solution of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipids in chloroform is purchased and aliquotted into clean glass vials in 20 mg per vial quantities for storage. A layer of nitrogen gas is added to each vial to prevent lipid oxidation, and the vials are sealed with caps and parafilm. The vials are stored in a -20° C freezer until use.
2. A single vial that contains an aliquot of 20 mg lipid in chloroform is used for each experiment. The contents of the vial are dried using a stream of nitrogen gas for 1 hour until no so....

Discussion

The current protocol describes the generation of unfolding curves of membrane-associated proteins and peptides that contain tryptophan residues. Here, it is assumed that the tryptophan fluorescence reflects whether the protein is folded and inserted into synthetic lipid vesicles, or unfolded in solution. Additional assumptions, such as two-state folding and linear dependence of free energy with denaturant concentration, are made in the current report; modification of these assumptions result in different equations.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMPC	Avanti Polar Lipid, Inc	850345C
Urea	MP Biomedicals	04821527
Potassium Phosphate Dibasic	Fisher Scientific	P288
Potassium Phosphate Monobasic	Fisher Scientific	P285