title: Direct analysis of protein sedimentation equilibrium in detergent solutions without density matching.
abstract: Characterizing membrane proteins by sedimentation equilibrium is challenging because detergents and/or lipid molecules, usually required for solubilization, form a complex with the protein. The most common way to overcome this problem is Tanford and Reynolds' density matching method, which eliminates the buoyant mass contributions of detergents/lipids by adjusting the solvent density with D2O/H2O mixtures to render either detergent or lipid molecules neutrally buoyant. Unfortunately, the method is practical only for detergent densities between 1.0 (H2O) and 1.1 (D2O) g ml(-1), excluding many of the more commonly used detergents for membrane protein studies. Here, we present a modern variant of Tanford and Reynolds' method that (1) is applicable to any detergent regardless of its specific density, (2) does not compromise accuracy and precision, and (3) provides additional information about the number of detergent molecules that are bound to each protein. The new method was applied successfully to Delta(1-43)A-I, an amino-terminal deletion mutant of human apolipoprotein A-I. Interestingly, we observed a significantly lower Delta(1-43)A-I/octyl-glucoside complex partial specific volume than that expected from volume additivity rules, indicative of specific protein-detergent interactions.
authors.affiliation: Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia PA 19104-6059, USA. dnoy@mail.med.upenn.edu
authors.forename: Dror
authors.initials: D
authors.lastname: Noy
authors.forename: Jennifer R
authors.initials: JR
authors.lastname: Calhoun
authors.forename: James D
authors.initials: JD
authors.lastname: Lear
mesh_terms: D016632:Apolipoprotein A-I / Q000032:analysis* / Q000378:metabolism; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D003902:Detergents; D006801:Humans; D014461:Ultracentrifugation / Q000379:methods
chemical_list: D016632:Apolipoprotein A-I; D003902:Detergents