Biophysical Society Thematic Meeting - October 13-15, 2015

Biophysics of Proteins at Surfaces: Assembly, Activation, Signaling Tuesday Speaker Abstracts

Quantifying the Membrane Assembly of Amphitropic Proteins by homo-FRET Analysis Ana M. Melo 1 , Aleksander Fedorov 1 , Manuel Prieto 1 , Ana Coutinho 1,2 . 1 Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal, 2 Faculdade de Ciências, Universidade de Lisboa, Lisbon, Portugal. Transient membrane recruitment of amphitropic proteins by anionic phospholipids is a common cellular mechanism involved in the regulation of membrane signal transduction. Here, we present a homo-FRET based method for the quantitative characterization of the oligomerization state of membrane-bound proteins involved in a three-state cooperative partition/oligomerization equilibria. We assume that monomeric proteins partition into the bilayer surface and reversibly assemble into homo k-mers. Using of a combination of steady-state and time-resolved fluorescence intensity and anisotropy measurements, this method was shown to be very robust in describing the electrostatic interaction of a model fluorescently-labelled amphitropic protein (Lz- A488) with anionic lipid membranes [1,2]. The pronounced decrease detected in the fluorescence anisotropy of membrane-bound Lz-A488, and therefore the extent of homo-FRET, always correlated with the system reaching a high surface coverage by the fluorescently-labeled protein at a low lipid-to-protein (L/P) molar ratio. Anisotropy decays of Lz-A488 samples prepared with variable fractional labeling (dye-to-protein molar ratios) further confirmed the occurrence of intra-oligomeric energy homo transfer-induced fluorescence depolarization. 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 ratio and protein fractional labeling) yielded that membrane-bound Lz-A488 self-assembled into oligomers with a stoichiometry of k= 6 ± 1. [1] Melo et al. 2013 J. Phys. Chem. 117: 2906−2917 (DOI: dx.doi.org/10.1021/jp310396v) [2] Melo et al. 2014 Phys.Chem.Chem.Phys. 16: 18105-18117 (DOI: 10.1039/c4cp00060a) This work was supported by FCT/Portugal (PTDC/BBB-BQB/2661/2012 and RECI/CTM- POL/0342/2012). A.M. Melo current address is Dept Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA.

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