Membrane proteins have evolved to function in a complex lipid environment. For example, many protein domains on the cytoplasmic side of the membrane are enriched in positively charged amino acid residues, which is likely because of the abundant anionic lipids in the inner leaflet of eukaryotic plasma membrane. These electrostatic lipid-protein interactions have been hypothesized to regulate structure and function at the membrane for many proteins. However, experimental investigations of these interactions are difficult because of the heterogeneous structure and dynamics in the plasma membrane. Here I will present an approach to directly measure lipid-protein interactions in supported lipid bilayers (SLBs) with time-resolved fluorescence spectroscopy. In the SLBs, the lipid of interest is tagged with a fluorescent probe in the acyl chain, leaving the anionic head group unmodified. The protein is labeled with an orthogonal fluorescent probe positioned away from the interaction interface. Pulsed-interleaved fluorescence cross-correlation spectroscopy (PIE-FCCS) is then used to monitor the mobility, concentration, and co-diffusion of the lipid and membrane-associated protein. With PIE-FCCS we can directly determine if and to what degree the lipids and proteins diffuse as a stable complex. This allows us to verify the interactions and determine side chain specificity through protein mutagenesis and lipid specificity by changing the SLB composition. This experimental data could provide valuable feedback in the development of improved computational models for lipid-protein interactions.

Anionic lipid binding to peripheral membrane proteins, what can fluorescence spectroscopy tell us?

2019 Spring National Meeting

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