The Chemistry of Life Processes Program in the Chemistry Division funds this work. Professor Yinan Wei from University of Kentucky investigates protein-protein interactions in the cell membrane that lead to the assembly of functional protein complexes. Selective-permeable membranes, which define the boundaries of individual cells and cellular organelles, are essential for energy production to sustain life. Membrane proteins are gates on these highly impermeable barriers, allowing selective exchange of substances and signals across the membrane. Understanding of the molecular recognition process leading to membrane protein structures is essential for improved understanding of protein evolution, function, and regulation. The aim this research is to establish a clear picture of the complexation of one model system, a multidrug efflux transporter, AcrB, in living cells and determine its stability in the cell membrane. This pursuit allows graduate and undergraduate students to acquire training in biochemistry and molecular biology research. This project also integrates into an outreach program to introduce science and science careers to girls ages 12-18. Dr. Wei is collaborates with the Hope Hill Youth Service (Mt Sterling, KY), a program for girls ages 12-18, who suffer from histories of abuse and neglect and have behavioral and emotional problems requiring extensive treatment and therapy. The Hope Hill Youth Service does not have STEM (Science, Technology, Engineering and Mathematics) education and so their students fall behind in math and science; the girls struggle to understand the opportunities that education can afford them, lacking dreams and motivation. The goals of these interactions are to broaden the knowledge of career opportunities for the girls.  In this study, AcrB is used as a model system and its oligomerization in both detergent micelles and lipid bilayers is quantitatively characterized. The kinetic and thermodynamic aspects of the trimer assembly process are monitored in cells and in reconstituted lipid bilayers using an array of assays and methods developed in preliminary studies. These include: a fluorescence resonance energy transfer (FRET)-based method that is compatible with the characterization of AcrB subunit interaction in a lipid bilayer environment, a fluorescence-based method to monitor protein unfolding, and a disulfide-trapping based method to characterize the structure of AcrB in the cell membrane in its native state. These assays are suitable for monitoring and characterizing AcrB oligomerization in both detergent micelles and lipid bilayers. Furthermore, a collection of AcrB mutants with decreased trimer affinities have been created. The following research objectives are pursued: 1) investigation of the process of AcrB trimer assembly in cells; 2) determination of AcrB trimer stability in the cell membrane; and 3) characterization of AcrB trimer dissociation in lipid bilayers. Outcomes from this project bring new insight into the kinetics and thermodynamics of assembly of a multi-span and multi-domain membrane protein trimer. The protocols and parameters developed in the research provide valuable tools and benchmarks for the research community, which lead the way to new initiatives in membrane protein research.
