Biophysical Society Thematic Meeting - October 13-15, 2015

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Biophysics of Proteins at Surfaces: Assembly, Activation, Signaling Tuesday Speaker Abstracts
Quantifying the Membrane Assembly of Amphitropic Proteins by homo-FRET Analysis
Ana M. Melo1 , Aleksander Fedorov1, Manuel Prieto 1, Ana Coutinho1,2.
1Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal, 2Faculdade 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: )
[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.