Workshop on AUC of Membrane Proteins
Presenters: Christine Ebel and
Caroline Mas, Institut de Biologie Structurale and ISBG, Grenoble, France, and
Karen Fleming, Johns Hopkins University, Baltimore, USA.
DescriptionIntegral 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
- How to determine the parameters needed for data analysis (partial specific volumes and increment of refractive indexes)
- Strategies for the design of the cells and experiments
- How to analyze the data using sedfit/sedphat/gussi programs
- How to evaluate size distribution and non-interacting species, sample homogeneity, protein association state, amounts of bound detergent, and the limits of these analysis.
- How to determine parameters needed for data analysis (partial specific volumes)
- Strategies for experimental design, including how to density match and what to do if density matching using water is not feasible
- How to globally analyze the data using WinNonlin
- How to evaluate molecular weight distributions for reversible equilibrium