Quantification of Protein-Lipid Selectivity using FRET: Application to the M13 Major Coat Protein

Abstract

Quantification of lipid selectivity by membrane proteins has been previously addressed mainly from electron spin resonance studies. We present here a new methodology for quantification of protein-lipid selectivity based on fluorescence resonance energy transfer. A mutant of M13 major coat protein was labeled with 7-diethylamino-3((4'iodoacetyl)amino)phenyl-4-methylcoumarin to be used as the donor in energy transfer studies. Phospholipids labeled with N-(7-nitro-2-1,3-benzoxadiazol-4-yl) were selected as the acceptors. The dependence of protein-lipid selectivity on both hydrophobic mismatch and headgroup family was determined. M13 major coat protein exhibited larger selectivity toward phospholipids which allow for a better hydrophobic matching. Increased selectivity was also observed for anionic phospholipids and the relative association constants agreed with the ones already presented in the literature and obtained through electron spin resonance studies. This result led us to conclude that fluorescence resonance energy transfer is a promising methodology in protein-lipid selectivity studies.

FIGURE 1

Molecular model for the FRET analysis ((A) side view; (B) top view). Protein-lipid organization presents a hexagonal geometry. Donor fluorophore from the mutant protein is located in the center of the bilayer, whereas the acceptors are distributed in the bilayer surface. Two different environments are available for the labeled lipids (acceptors), the annular shell surrounding the protein and the bulk lipid. Energy transfer to acceptors in direct contact with the protein has a rate coefficient dependent on the distance between donor and annular acceptor (Eq. 8). Energy transfer toward acceptors in the bulk lipid is given by Eq. 11 (see text for details).

FIGURE 2

Corrected emission spectrum of DCIA-labeled M13 major coat protein (—), and corrected excitation spectrum of NBD-derivatized phospholipid ().

FIGURE 3

Donor (DCIA-labeled protein) fluorescence quenching by energy transfer acceptor ((18:1)₂-PE-NBD) in pure phosphatidylcholine bilayers with different hydrophobic thickness. (•), Experimental energy transfer efficiencies; (—), theoretical simulations obtained from the annular model for protein-lipid interaction using the fitted K_S; and (), simulations for random distribution of acceptors (K_S = 1.0). (A) Labeled protein incorporated in DOPC (fitted K_S = 1.4); (B) labeled protein incorporated in DMoPC (fitted K_S = 2.9); and (C) labeled protein incorporated in DEuPC (fitted K_S = 2.1).

FIGURE 4

Donor (DCIA-labeled protein) fluorescence quenching by energy transfer acceptor (18:1-(12:0-NBD)-PX), where X stands for the different headgroup structures, in pure bilayers of DOPC. (•), Experimental energy transfer efficiencies; (—), theoretical simulations obtained from the annular model for protein-lipid interaction using the fitted K_S; and (), simulations for random distribution of acceptors (K_S = 1.0). (A) PC-labeled phospholipid (fitted K_S = 2.0); (B) PE-labeled phospholipid (fitted K_S = 2.0); (C) PG-labeled phospholipid (fitted K_S = 2.3); (D) PS-labeled phospholipid (fitted K_S = 2.7); and (E) PA-labeled phospholipid (fitted K_S = 3).