title: Thermodynamics of membrane protein folding measured by fluorescence spectroscopy.
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, Δ G°(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.
authors.affiliation: Chemistry and Biochemistry, University of California San Diego - UCSD, USA.
authors.forename: Diana E
authors.initials: DE
authors.lastname: Schlamadinger
authors.forename: Judy E
authors.initials: JE
authors.lastname: Kim
mesh_terms: D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013816:Thermodynamics
chemical_list: D008565:Membrane Proteins