Depth-Dependent Heterogeneity in Membranes by Fluorescence Lifetime Distribution Analysis

Biological membranes display considerable anisotropy due to differences in composition, physical characteristics, and packing of membrane components. In this Letter, we have demonstrated the environmental heterogeneity along the bilayer normal in a depth-dependent manner using a number of anthroyloxy fatty acid probes. We employed fluorescence lifetime distribution analysis utilizing the maximum entropy method (MEM) to assess heterogeneity. Our results show that the fluorescence lifetime heterogeneity varies considerably depending on fluorophore location along the membrane normal (depth), and it is the result of the anisotropic environmental heterogeneity along the bilayer normal. Environmental heterogeneity is reduced as the reporter group is moved from the membrane interface to a deeper hydrocarbon region. To the best of our knowledge, our results constitute the first experimental demonstration of anisotropic heterogeneity in bilayers. We conclude that such graded environmental heterogeneity represents an intrinsic characteristics of the membrane bilayer and envisage that it has a role in the conformation and orientation of membrane proteins and their function.