Recent Developments in the Study of the Behavior of Fluorescent Membrane Probes in Lipid Bilayers: Molecular Dynamics Approach

Abstract

The molecular level organization of biomembranes and the establishment of structure/function relationships of membrane-active biomolecules are topics of crucial importance for the understanding of many phenomena in cell biophysics and biochemistry. This poses challenging problems that most frequently require the use of advanced experimental techniques (Gennis, 1989), among which fluorescence stands out as one of the most powerful and commonly used due to its sensitivity and versatility (Lakowicz, 2006; Royer & Scarlata, 2008). The sub-nanosecond time resolution of fluorescence allows following the kinetics of both fast (fluorophore rotation, conformational changes) and relatively slow (translocation, hindered diffusion) processes, and its extremely high sensitivity (as evidenced in techniques such as single-fluorophore imaging and fluorescence correlation spectroscopy) is almost unrivaled. But, perhaps the most salient feature of fluorescence spectroscopy is its high versatility which allows, through different parameters, the retrieval of complementary molecular information on the system under study. Spectral and fluorescence lifetime/quantum yield variations are informative regarding the polarity and/or solvent accessibility of the fluorophore microenvironment and the extent of partition of a fluorophore-bearing molecule between the aqueous and membrane media, or between coexisting membrane phases or domains. Fluorescence quenching, depending on the underlying interaction mechanism and on the experimental design, can be used to study molecular aggregation, lateral diffusion, compartmentalization, or transverse location in the bilayer. Fluorescence polarization is used to measure the viscosity of the microenvironment and the kinetics of fluorophore rotation. Forster resonance energy transfer (FRET) is useful in many common situations, such as detection and characterization of membrane heterogeneity, determination of the transverse location of protein fluorophores, detection