Biophysical Society Thematic Meeting | Ascona, Switzerland

Liposomes, Exosomes, and Virosomes: From Modeling Complex Membrane Processes to Medical Diagnostics and Drug Delivery

Poster Abstracts

57-POS Board 29 Probing Lipid-Protein Interactions in Situ with Single-Molecule Sensitivity Xiaojun Shi , Shaun M. Christie, Grant T. Gilmore, Adam W. Smith. the University of Akron, Akron, OH, USA.

Proteins interact with PIP lipids in the plasma membrane (PM). These interactions are ubiquitous in cell signaling pathways and regulate a wide variety of cellular processes. The kinetic and thermodynamic details of these interactions, however, are difficult to probe directly in biological membranes. Here we investigate the behavior of PIP lipids in supported lipid bilayers (SLBs) in response to protein binding with two single-molecule time-resolved fluorescence techniques: pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) and single particle tracking (SPT). PIP lipids are introduced to the top leaflet of the supported lipid bilayers using a novel method recently developed in our group. In this way we can make background-free SLBs with a range of PIP concentrations that are suitable for both SPT and PIE-FCCS measurements of lipid-protein interaction at the membrane interface. We probe the interactions of PIP with adsorbed proteins by characterizing changes in PIP mobility caused by protein binding. We then measure the co-diffusion of proteins with PIP lipids as direct evidence of lipid- protein complexation. PIE-FCCS allows us to characterize the size and stability of the lipid- protein complex, and the dependency on charge interactions in different experimental conditions (such as charge density of the lipid head group and solvent conditions). We will report on recent work with model peptides and as well as with the kinase domain of EphA2. Interactions between the cytoplasmic domain of receptor tyrosine kinases and the inner leaflet of the PM have been implicated in receptor autoinhibition. Our assay is an ideal platform to investigate the affinity and specificity of lipid-protein interactions in a uniquely quantitative way.

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