We measured the lateral mobility of integral membrane proteins reconstituted in giant unilamellar vesicles (GUVs), using fluorescence correlation spectroscopy. Receptor, channel, and transporter proteins with 1-36 transmembrane segments (lateral radii ranging from 0.5 to 4 nm) and a alpha-helical peptide (radius of 0.5 nm) were fluorescently labeled and incorporated into GUVs. At low protein-to-lipid ratios (i.e., 10-100 proteins per microm(2) of membrane surface), the diffusion coefficient D displayed a weak dependence on the hydrodynamic radius (R) of the proteins [D scaled with ln(1/R)], consistent with the Saffman-Delbruck model. At higher protein-to lipid ratios (up to 3000 microm(-2)), the lateral diffusion coefficient of the molecules decreased linearly with increasing the protein concentration in the membrane. The implications of our findings for protein mobility in biological membranes (protein crowding of approximately 25,000 microm(-2)) and use of diffusion measurements for protein geometry (size, oligomerization) determinations are discussed.

We investigated the effect of amino acid composition and hydrophobic length of alpha-helical transmembrane peptides and the role of electrostatic interactions on the lateral diffusion of the peptides in lipid membranes. Model peptides of varying length and composition, and either tryptophans or lysines as flanking residues, were synthesized. The peptides were labeled with the fluorescent label Alexa Fluor 488 and incorporated into phospholipid bilayers of different hydrophobic thickness and composition. Giant unilamellar vesicles were formed by electroformation, and the lateral diffusion of the transmembrane peptides (and lipids) was determined by fluorescence correlation spectroscopy. In addition, we performed coarse-grained molecular-dynamics simulations of single peptides of different hydrophobic lengths embedded in planar membranes of different thicknesses. Both the experimental and simulation results indicate that lateral diffusion is sensitive to membrane thickness between the peptides and surrounding lipids. We did not observe a difference in the lateral diffusion of the peptides with respect to the presence of tryptophans or lysines as flanking residues. The specific lipid headgroup composition of the membrane has a much less pronounced impact on the diffusion of the peptides than does the hydrophobic thickness.

Microcavity supported lipid bilayers (MSLB) are contact-free membranes suspended across aqueousfilled pores that maintain the lipid bilayer in a highly fluidic state and free from frictional interactions with substrate. Such platforms offer the prospect of liposome-like fluidity with the compositional versatility and addressability of supported lipid bilayers and thus offer significant opportunity for modelling membrane asymmetry, protein-membrane interactions and aggregation at the membrane interface. Herein, we evaluate their performance by studying the effect of transmembrane lipid asymmetry on lipid diffusivity, membrane viscosity and cholera toxin-ganglioside recognition across six symmetric and asymmetric membranes including binary compositions containing both fluid and gel phase, and ternary phase separated membrane compositions. Fluorescence lifetime correlation spectroscopy (FLCS) was used to determine the lateral mobility of lipid and protein, and electrochemical impedance spectroscopy (EIS) enabled detection of protein-membrane assembly over the nanomolar range. Transmembrane leaflet asymmetry was observed to have profound impact on membrane electrochemical resistance where the resistance of a ternary symmetric phase separated bilayer was found to be at least 2.6 times higher than the asymmetric bilayer with analogous composition at the distal leaflet but where the lower leaflet comprised only 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). Similarly, the diffusion coefficient for MSLBs was observed to be 2.5 fold faster for asymmetric MSLBs where the lower leaflet is DOPC alone. Our results demonstrate that interplay of lipid packing across both membrane leaflets and concentration of GM1 both affect the extent of cholera toxin aggregation and consequent diffusion of the cholera-GM1 aggregates. Given that true biomembranes are both fluidic and asymmetric, MSLBs offer the opportunity for building greater biomimicry into biophysical models and the approach described demonstrates the value of MSLBs in studying aggregation and membrane associated multivalent interactions prevalent in many carbohydrates mediated processes.

Biological membranes are composed of a large number lipid species differing in hydrophobic length, degree of saturation, and charge and size of the headgroup. We now present data on the effect of hydrocarbon chain length of the lipids and headgroup composition on the lateral mobility of the proteins in model membranes. The trimeric glutamate transporter (GltT) and the monomeric lactose transporter (LacY) were reconstituted in giant unilamellar vesicles composed of unsaturated phosphocholine lipids of varying acyl chain length (14-22 carbon atoms) and various ratios of DOPE/DOPG/DOPC lipids. The lateral mobility of the proteins and of a fluorescent lipid analog was determined as a function of the hydrophobic thickness of the bilayer (h) and lipid composition, using fluorescence correlation spectroscopy. The diffusion coefficient of LacY decreased with increasing thickness of the bilayer, in accordance with the continuum hydrodynamic model of Saffman-Delbrück. For GltT, the mobility had its maximum at diC18:1 PC, which is close to the hydrophobic thickness of the bilayer in vivo. The lateral mobility decreased linearly with the concentration of DOPE but was not affected by the fraction of anionic lipids from DOPG. The addition of DOPG and DOPE did not affect the activity of GltT. We conclude that the hydrophobic thickness of the bilayer is a major determinant of molecule diffusion in membranes, but protein-specific properties may lead to deviations from the Saffman-Delbrück model.

Many drugs have intracellular or membrane-associated targets thus understanding their interaction with the cell membrane is of value in drug development. Cell-free tools used to predict membrane interactions should replicate the molecular organization of the membrane. Microcavity array supported lipid bilayer (MSLB) platform are versatile biophysical models of the cell membrane that combine liposome-like membrane fluidity with stability and addressability. We used an MSLB herein to interrogate drugmembrane interactions across seven drugs from different classes, including non-steroidal antiinflammatories; Ibuprofen (Ibu) and Diclofenac (Dic), antibiotics; Rifampicin (Rif), Levofloxacin (Levo) and Pefloxacin (Pef), and bisphosphonates; Alendronate (Ale) and Clodronate (Clo). Fluorescence lifetime correlation spectroscopy (FLCS) and electrochemical impedance spectroscopy (EIS) were used to evaluate the impact of drug on DOPC and binary bilayers over physiologically relevant drug concentrations. Whereas FLCS data revealed Ibu, Levo, Pef, Ale and Clo had no impact on lipid lateral