Abstract

In this review, we present the progress in development and application of fluorescence spectroscopy approaches for studying posttranslational insertion of membrane proteins into lipid bilayers. Various methods utilizing environment-sensitive probes, FRET, FCS and fluorescent quenching for structural and thermodynamic characterization of protein-lipid interactions are discussed. In addition, we demonstrate the use of fluorinated surfactants, which do not interact with the lipid bilayer, but can chaperone membrane protein insertion. An FCS-based protocol is presented for the determination of the free energy of transmembrane insertion of membrane proteins and hydrophobic peptides, chaperoned by the surfactants. We also describe the application of steady-state and time-resolved fluorescence quenching by lipid-attached quenchers to characterize membrane protein immersion into the lipid bilayer. Finally, we illustrate the use of the entire battery of spectroscopic approaches to characterize structural, kinetic and thermodynamic properties of the pH-triggered insertion/refolding pathway of the diphtheria toxin translocation domain.