Sedimentation velocity to characterize surfactants and solubilized membrane proteins.
Analytical ultracentrifugation sedimentation velocity, which combines the separation of the macromolecules and the analysis of their transportation to reach a rigorous thermodynamics study offers a robust tool for characterizing the homogeneity and association state of membrane protein. Samples of solubilized membrane proteins are indeed complex multi-component systems where detergent micelles and protein-detergent complexes coexist in solution, with associated lipids in variable amounts. We present here the sedimentation velocity theoretical background, the principle of the data analysis and the interpretation relevant for the study of membrane proteins. The results section presents examples and refers to published work. High resolution particle distribution are obtained from measurements in absorbance and interference, which permits the characterization of protein-detergent complexes-in terms of association state and bound detergents/lipids, even in heterogeneous samples, and of surfactants. We emphasize the precaution to be taken before the analysis, and the limits of the analysis. We show how the sedimentation velocity performed in H(2)O and D(2)O solvents may help to acertain the association state of solubilized membrane proteins. We discuss the complementarity with sedimentation equilibrium, density measurement, and size exclusion chromatography combined if necessary with the use of radiolabelled detergent or light scattering detection.
CEA, Institut de Biologie Structurale, IBS, Grenoble, 41 rue Jules Horowitz, F-38027 Grenoble, France. christine.ebel@ibs.fr
Christine
C
Ebel
D003902:Detergents / Q000737:chemistry; D017666:Deuterium Oxide / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry*; D012995:Solubility; D013816:Thermodynamics; D014461:Ultracentrifugation / Q000379:methods*; D014867:Water / Q000737:chemistry
D003902:Detergents; D008565:Membrane Proteins; D014867:Water; D017666:Deuterium Oxide