Phospholipid packing asymmetry in curved membranes detected by fluorescence spectroscopy

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Abstract
There are distinct differences in the molecular packing of phospholipid molecules in the inner and outer membrane monolayers of small lipid vesicles; a small radium of curvature imparts an asymmetry to the interface between these two monolayers. I have used an amphiphilic fluorescent probe, N-[5-(dimethylamino)naphthalenyl-1-sulfonyl]glycine (dansylglycine), to determine if this asymmetry in molecular packing leads to the existence of different environments for fluorescent probes resident in the membrane. Dansylglycine is highly sensitive to the dielectric constant of its environment, and the fluorescence signal from membrane-bound dye is distant from that in the aqueous medium. When dansylglycine is first mixed with vesicles, it rapidly partitions into the outer monolayer; the subsequent movement of dye into the inner monolayer is much slower. Because of the time lag between the initial partitioning and the subsequent translocation, it is possile to measure the emission spectrum from membrane-bound dye before and after translocation, thus distinguishing the two potential environments for dansylglycine molecules. In the outer membrane monolayer of small dipalmitoylphosphatidylcholine vesicles, dye fluorescence emission is maximal at 530 nm, corresponding to a dielectric constant of 7 for the medium surrounding the fluorophore. For dye in the inner monolayer, emission is maximal at 519 nm, corresponding to a dielectric constant of 4.7. The results suggest that water molecules are excluded more efficiently from the dye binding sites of the inner membrane monolayer than they are from those of the outer monolayer. When similar measurements were made on vesicles with a very large radius of curvature, there was no detectable difference in the wavelength of maximal emission from dye located in the outer or inner monolayers, both locations giving rise to emission at approximately 535 nm, corresponding to a dielectric constant of 10. Thus, it seems that in small lipid vesicles the phospholipid packing geometry of the outer membrane monolayer closely parallels that of a planar bilary. In contrast, the inner monolayer appears to be subjected to packing constraints unique to systems with small radius of curvature.