Membrane Proteins

Workshop on AUC of Membrane Proteins

Presenter: Karen Fleming, T.C. Jenkins Dep. of Biophysics, Johns Hopkins University, MD USA, and Christine Ebel and Aline Le Roy, IBS, CEA, CNRS, Univ. Grenoble Alpes, France


Integral membrane proteins (MPs) are physiologically embedded in a lipid bilayer, which provides a hydrophobic environment compatible with their nonpolar, transmembrane surfaces. Both structural and solution studies of MPs often involve their extraction, solubilization, purification, and characterization in a wide variety of detergent micelles. In this workshop we will describe the AUC characterization and analysis of detergent solubilized membrane proteins. Parameters that can be evaluated include MP homogeneity, protein molar mass, bound detergent, and protein-protein association constants.

The AUC challenge of solubilized membrane protein samples is principally that detergent-solubilized membrane proteins represent a multicomponent system, in which different components (protein, detergent, lipid, water?) interact to form the different types of sedimenting particles (species). The samples are therefore necessarily polydisperse, principally because of the presence of free detergent micelles in solution. Depending on the specific conditions, this complexity can be disentangled by taking advantage of several key distinctions between protein and detergent properties. For example, detergents and proteins have distinct optical properties (different extinction coefficients and increments of refractive index), and different buoyant properties (partial specific volumes). Moreover, membrane protein-protein interactions (as protein stability) can significantly depend on the type and concentration of detergent. In addition to a presentation of the theoretical considerations and mathematical formalism, we will discuss practical implementation and analysis of both sedimentation velocity and sedimentation equilibrium experiments and analysis of membrane protein/detergent solutions. Topics to be covered include the following:

Sedimentation Velocity

Sedimentation Equilibrium

This workshop will be presented twice on Sunday during session 1, with a repeat on Sunday during session 2.