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Liposomes, Exosomes, and Virosomes: From Modeling Complex
Membrane Processes to Medical Diagnostics and Drug Delivery
Poster Abstracts
113
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.