Development of a Platform Enabling Analysis of Membrane Protein Interactions

Proteins embedded within or associated with cellular membranes make up 30% of all proteins. These cellular gatekeepers are critical in biological functions, including transport in and out of cells, recognition and transduction of information, energy production and communication amongst cells. Despite the importance of membrane proteins, understanding of the interactions and functions of the membrane proteome is still greatly hampered by technical challenges; there is an urgent need for innovative approaches to uncover how membrane proteins interact with one another and with their binding partners in the native membrane environment. This project will develop a versatile experimental platform that provides maps of protein-protein and protein-membrane interactions. Under-represented minorities will receive STEM education and training at undergraduate graduate level. The proposal will also provide video instructions for high school students to attract students in STEM fields.

This collaborative research will establish a versatile experimental platform that provides information on protein-protein and protein-membrane assemblies in Nanodiscs, which represent a native-like model membrane system. The goals of this research are to establish a versatile experimental platform that provides vector maps of protein-protein and protein-membrane assemblies. Key cornerstones of the approach are the use of lipid bilayer nanodiscs (NDs) as a native-like membrane platform and biophysical approaches that rely on strategically positioned lanthanide-binding tags (LBTs). The LBTs are dual-function probes that insert atoms for physical measurements enabling the construction of three-dimensional vector maps of protein assemblies relative to the membrane plane. Measurements will be accomplished via small-angle X-ray scattering (SAXS) and anomalous SAXS (ASAXS) to provide distances between lanthanides and center of mass and inter-lanthanide distances. The construction of vector maps will allow accurate measurement of the response of proteins to the presence and absence of binding partners, dynamic protein modifications, and indeed the composition of the membrane itself-thus defining the critical determinants of both assembly and function. This project is supported by the Molecular Biophysics Cluster of the Molecular and Cellular Biosciences Division in the Directorate for Biological Sciences.