Förster resonance energy transfer (FRET) is a photophysical process by which a donor (D) molecule in an electronic excited state transfers its excitation energy to a second species, the acceptor (A). Since FRET efficiency depends on D-A separation, the measurement of donor fluorescence in presence and absence of the acceptor allows determination of this distance, and therefore FRET has been extensively used as a "spectroscopic ruler". In membranes, interpretation of FRET is more complex, since one D may be surrounded by many A molecules. Such is the case encountered with membrane proteins and lipids in the bilayer. This paper reviews the application of a model built to analyze FRET data between a single tryptophan mutant of the transmembrane protein lactose permease (W151/C154G of LacY), the sugar/H(+) symporter from Escherichia coli, and different pyrene-labeled phospholipids. Several variables of the system with biological implication have been investigated: The selectivity of LacY for different species of phospholipids, the enhancement of the sensitivity of the FRET modeling, and the mutation of a particular aminoacid (D68C) of the protein. The results obtained support: (i) Preference of LacY for phosphatidylethanolamine (PE) over phosphatidylglycerol (PG); (ii) affinity of LacY for fluid (L(α)) phases; and (iii) importance of the aspartic acid in position 68 in the sequence of LacY regarding the interaction with the phospholipid environment. Besides, by exploring the enhancement of the sensitivity by using pure lipid matrices with higher mole fractions of labelled-phospholipid, the dependence on acyl chain composition is unveiled.
Keywords: FRET; lactose permease; lipid–protein interactions.