title: Measuring membrane protein stability under native conditions.
abstract: The thermodynamic stability of proteins is typically measured at high denaturant concentrations and then extrapolated back to zero denaturant conditions to obtain unfolding free energies under native conditions. For membrane proteins, the extrapolations are fraught with considerable uncertainty as the denaturants may have complex effects on the membrane or micellar structure. We therefore sought to measure stability under native conditions, using a method that does not perturb the properties of the membrane or membrane mimetics. We use a technique called steric trapping to measure the thermodynamic stability of bacteriorhodopsin in bicelles and micelles. We find that bacteriorhodopsin has a high thermodynamic stability, with an unfolding free energy of ∼11 kcal/mol in dimyristoyl phosphatidylcholine bicelles. Nevertheless, the stability is much lower than predicted by extrapolation of measurements made at high denaturant concentrations. We investigated the discrepancy and found that unfolding free energy is not linear with denaturant concentration. Apparently, long extrapolations of helical membrane protein unfolding free energies must be treated with caution. Steric trapping, however, provides a method for making these measurements.
authors.affiliation: Department of Chemistry and Biochemistry, UCLA-DOE Institute for Genomics and Proteomics, Molecular Biology Institute, University of California, Los Angeles, CA 90095.
authors.forename: Yu-Chu
authors.initials: YC
authors.lastname: Chang
authors.forename: James U
authors.initials: JU
authors.lastname: Bowie
mesh_terms: D001436:Bacteriorhodopsins / Q000737:chemistry*; D001710:Biotin; D019921:Biotinylation; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry*; D019617:Halobacterium salinarum / Q000737:chemistry; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry*; D008823:Micelles; D011489:Protein Denaturation; D017510:Protein Folding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D013696:Temperature; D013816:Thermodynamics
chemical_list: D008051:Lipid Bilayers; D008565:Membrane Proteins; D008823:Micelles; D001436:Bacteriorhodopsins; D001710:Biotin; D004134:Dimyristoylphosphatidylcholine
keywords: membrane protein folding; steric trap