Oligomer Stoichiometry of Membrane-Bound Proteins Involved in a Cooperative Partition Equilibrium: A Homo-FRET Study

Abstract

An analytical framework that uses energy homo transfer to directly probe quantitatively the oligomerization state of membrane-bound proteins engaged in a three-state cooperative partition is presented [1]. It was assumed that monomeric proteins partition into the bilayer surface and reversibly assemble into oligomers with k subunits [2]. A general equation relating the overall steady-state fluorescence anisotropy of the sample to its fractional labeling was derived by considering explicitly that the anisotropy of mixed oligomers containing i-labeled monomers is inversely proportional to the number of labeled subunits per oligomer (Runnels and Scarlata limit). This method was very robust in describing the electrostatic interaction of Alexa 488 fluorescently-labeled lysozyme (Lz-A488) with phosphatidylserine-containing membranes. The pronounced decrease detected in the fluorescence anisotropy of Lz-A488 always correlated with the system reaching a high membrane surface density of the protein (low L/P molar ratio). The occurrence of energy homo transfer-induced fluorescence depolarization was further confirmed by measuring the anisotropy decays of Lz-A488 under these conditions. A global analysis of the steady-state anisotropy data obtained under a wide range of experimental conditions (variable anionic lipid content of the liposomes, L/P molar ratios and protein fractional labeling) confirmed that membrane-bound Lz-A488 assembled into oligomeric complexes, possibly with a stoichiometry of k= 6 ± 1. This study illustrates that even in the presence of a coupled partition/oligomerization equilibria, steady-state anisotropy measurements can be used to monitor the self-assembly of membrane-bound proteins.