Biophysical Analysis of Phosphatidylserine-Binding Proteins Lactadherin C2 and the Ebola Virus Matrix Protein VP40

Characterization of lipid-protein interactions are of paramount importance to fundamental biophysics as well as in understanding potential therapeutic targets.Approximately 30% of cellular proteins are membrane-integrated or membrane-adjacent, leaving a vast field that has been primarily unexplored namely due to difficulty of experimentation and lack of quantitative techniques.Here, I characterize the biophysical interactions of two membrane-affiliated proteins, the biosensor LactC2, and the Ebola virus matrix protein VP40, through a series of Surface Plasmon Resonance (SPR), cell culture, mutagenesis, and lipidomic analysis experiments.

The biosensor LactC2 has been characterized as a tool to detect levels of phosphatidylserine (PS), an anionic, inner-leaflet plasma membrane lipid.This tool has been used both in cell culture experiments as well as in vitro binding studies by a number of labs.However, this protein had not been explicitly characterized vis a vis its particular lipid environment.SPR experiments have helped fill the void in the field regarding these interactions, noting particularly that cholesterol and membrane fluidity impact LactC2 affinity to membranes.

The Ebola virus matrix protein VP40 is the primary driving factor in Ebola (EBOV) assembly and egress.This protein has only very recently been able to be characterized extensively thanks to crystallographic data that provided foundational structural knowledge onto which functional analysis could be built.Prior work on VP40-lipid interactions demonstrated the necessity of PS for proper VP40 cellular function, but had not further characterized this biophysical interaction. Mutagenesis studies of VP40 implicate a highly conserved C-terminal domain that is necessary for proper membrane binding and function within cells.Lipidomic analysis of cells transfected with VP40 and their progeny Virus Like Particles (VLPs) preliminarily implicate the preference for saturated species of phosphatidylserine within packaged virus particles.The data obtained in these studies have enhanced our understanding of protein-lipid interactions for both the biosensor LactC2 as well as the Ebola Virus Matrix Protein VP40.