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Project Summary
Life on earth began with the development of selective-permeable membranes, which define the boundaries of individual cells and cellular organelles, and 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. The majority of membrane proteins function as oligomers, forming transient or long-lasting interactions in the membrane. However, it is not yet clear how protein-protein interaction is established during oligomer assembly in cell membrane, especially for homo-oligomers containing more than two protomers. Understanding of the molecular recognition process leading to membrane protein oligomerization and oligomer stability in cell membrane is essential to research in areas of protein evolution, function, and regulation. The long term goal of our research is to understand how proteins oligomerize in the cell membrane and how oligomerization determines protein function. As a step toward the long-term goal, the aim of the proposed project is to establish a clear picture of the oligomerization process of AcrB in living cells and determine the oligomer stability in cell membrane.
Intellectual Merit: AcrB is a multidrug efflux transporter in E. coli. It contains both a periplasmic domain and a transmembrane domain, with twelve transmembrane helices in each protomer. AcrB exists and functions exclusively as a homo-trimer. In the past several years, we have developed AcrB into a suitable model system for the study of membrane protein oligomerization. We have developed a FRET-based method that is compatible with the characterization of AcrB subunit interaction in a lipid bilayer environment, a fluorescence-based method to monitor protomer unfolding, and a disulfide-trapping based method to characterize the structure of AcrB in the cell membrane in its native state. These assays enable us to monitor and characterize AcrB oligomerization in both detergent micelles and lipid bilayers. Furthermore, we have created a collection of AcrB mutants with decreased trimer affinities. Built on top of our preliminary studies, we will pursue the following research objectives: 1) To investigate the process of AcrB trimer assembly in living cells; 2) To determine AcrB trimer stability in cell membrane; and 3) To characterize AcrB trimer dissociation in lipid bilayer. Outcomes from this project will bring new insight into the kinetics and thermodynamics of assembly of a multi-span and multi-domain membrane protein trimer. The protocols and parameters that will be developed in the proposed research will provide valuable tools and benchmarks for our future studies as well as other investigators studying protein-protein interaction in the cell membrane, which will lead the way to new initiatives in membrane protein research.
Broader Impacts: AcrB belongs to the resistance-nodulation-cell-division (RND) family of efflux pumps, which are conserved in all Gram-negative bacteria and play major roles in both intrinsic and acquired multi-drug resistance. We expect discoveries made from studying AcrB will be applicable to other proteins in the RND family as well, which may eventually lead to the design of novel strategies to block the assembly of these trimeric proteins. The proposed research will provide an opportunity for students of all levels to conduct modern biochemistry and molecular biology research, including 3 to 4 graduate students and 4 to 5 undergraduate and high school students. The PI will provide an opportunity for students to work in a multidisciplinary and multicultural environment, preparing them for an increasingly international and global working environment. The scientific discoveries resulting from the proposed research will be disseminated to the membrane protein and protein folding research communities through publishing papers in peer reviewed journals and presenting at research conferences.
Status | Finished |
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Effective start/end date | 8/1/17 → 12/31/21 |
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