title: Determination of membrane protein stability via thermodynamic coupling of folding to thiol-disulfide interchange.
abstract: Although progress has been made in understanding the thermodynamic stability of water-soluble proteins, our understanding of the folding of membrane proteins is at a relatively primitive level. A major obstacle to understanding the folding of membrane proteins is the discovery of systems in which the folding is in thermodynamic equilibrium, and the development of methods to quantitatively assess this equilibrium in micelles and bilayers. Here, we describe the application of disulfide cross-linking to quantitatively measure the thermodynamics of membrane protein association in detergent micelles. The method involves initiating disulfide cross-linking of a protein under reversible redox conditions in a thiol-disulfide buffer and quantitative assessment of the extent of cross-linking at equilibrium. The 19-46 alpha-helical transmembrane segment of the M2 protein from the influenza A virus was used as a model membrane protein system for this study. Previously it has been shown that transmembrane peptides from this protein specifically self-assemble into tetramers that retain the ability to bind to the drug amantadine. We used thiol-disulfide exchange to quantitatively measure the tetramerization equilibrium of this transmembrane protein in dodecylphosphocholine (DPC) detergent micelles. The association constants obtained agree remarkably well with those derived from analytical ultracentrifugation studies. The experimental method established herein should provide a broadly applicable tool for thermodynamic studies of folding, oligomerization and protein-protein interactions of membrane proteins.
authors.affiliation: Department of Biochemistry & Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6059, USA.
authors.forename: Lidia
authors.initials: L
authors.lastname: Cristian
authors.forename: James D
authors.initials: JD
authors.lastname: Lear
authors.forename: William F
authors.initials: WF
authors.lastname: DeGrado
mesh_terms: D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008823:Micelles; D010084:Oxidation-Reduction; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism*; D013816:Thermodynamics; D014461:Ultracentrifugation
chemical_list: D004220:Disulfides; D008565:Membrane Proteins; D008823:Micelles; D013438:Sulfhydryl Compounds; D005978:Glutathione