Abstract

Determination of the assembly state(s) of a membrane protein poses a difficult analytical problem. A powerful technique that is well adapted to meet this challenge is solution interaction analysis using analytical ultracentrifugation. Due to their hydrophobic nature, membrane proteins require detergent (or lipid) for solubilization in solution. Thus, a fundamental obstacle to be overcome in determining the stoichiometry of membrane proteins is that the mass of any bound detergent molecules will contribute to the experimentally determined apparent molecular weight. Judicious choice of detergent and solvent conditions surmounts this obstacle and facilitates evaluation of the mass of only the protein component in a protein–detergent complex. Employing this experimental strategy, sedimentation equilibrium approaches were used to determine the association states of the glycophorin A transmembrane (GpATM) dimer in solutions of two detergents, C12E8 and C8E5. Analysis of sedimentation equilibrium data collected in C8E5 micelles allowed estimation of a monomer/dimer equilibrium constant from which the free energy of dimerization could be calculated. Equilibrium constants were also determined for GpATM mutants that had previously been shown in a qualitative manner to diminish dimerization. Thus, the earlier mutational sensitivity results are placed on a quantitative, relative scale of interaction by ultracentrifuge experiments. This bulletin will focus on explaining the theoretical and practical considerations for making these types of experimental measurements on membrane proteins.

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