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Unspecific membrane protein–lipid recognition: combination of AFM imaging, force spectroscopy, DSC and FRET measurements
Author(s) -
Borrell Jordi H.,
Montero M. Teresa,
Morros Antoni,
Domènech Òscar
Publication year - 2015
Publication title -
journal of molecular recognition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.401
H-Index - 79
eISSN - 1099-1352
pISSN - 0952-3499
DOI - 10.1002/jmr.2483
Subject(s) - atomic force microscopy , förster resonance energy transfer , force spectroscopy , biophysics , chemistry , lipid bilayer , membrane , nanotechnology , materials science , biochemistry , fluorescence , biology , optics , physics
In this work, we will describe in quantitative terms the unspecific recognition between lactose permease (LacY) of Escherichia coli , a polytopic model membrane protein, and one of the main components of the inner membrane of this bacterium. Supported lipid bilayers of 1‐palmitoyl‐2‐oleoyl‐ sn ‐glycero‐3‐phosphoethanolamine (POPE) and 1‐palmitoyl‐2‐oleoyl‐ sn ‐glycero‐3‐phosphoglycerol (POPG) (3:1, mol/mol) in the presence of Ca 2+ display lateral phase segregation that can be distinguished by atomic force microscopy (AFM) as well as force spectroscopy. LacY shows preference for fluid ( L α ) phases when it is reconstituted in POPE : POPG (3:1, mol/mol) proteoliposomes at a lipid‐to‐protein ratio of 40. When the lipid‐to‐protein ratio is decreased down to 0.5, two domains can be distinguished by AFM. While the upper domain is formed by self‐segregated units of LacY, the lower domain is constituted only by phospholipids in gel ( L β ) phase. On the one hand, classical differential scanning calorimetry (DSC) measurements evidenced the segregation of a population of phospholipids and point to the existence of a boundary region at the lipid–protein interface. On the other hand, Förster Resonance Energy Transfer (FRET) measurements in solution evidenced that POPE is selectively recognized by LacY. A binary pseudophase diagram of POPE : POPG built from AFM observations enables to calculate the composition of the fluid phase where LacY is inserted. These results are consistent with a model where POPE constitutes the main component of the lipid–LacY interface segregated from the fluid bulk phase where POPG predominates. Copyright © 2015 John Wiley & Sons, Ltd.