Protein Activity and Oligomer Stability in Cell Membrane

  • Wei, Yinan (PI)

Grants and Contracts Details


Intellectual Merit: The selective permeability of cell membranes, which is essential for all life forms as we know, is conferred by membrane proteins. Approximately 80% of membrane proteins with known structures exist as oligomers when crystallized, indicating a large portion of proteins function as oligomers in cell membrane. However, it is not yet clear how proteinprotein interaction in cell membrane is established during oligomerization and how stabilities of such interactions determine function. A thorough understanding of this fundamental aspect of biological science will have an immense impact on many research areas including the biogenesis of cell membrane, establishment of homeostasis, signal transduction and material transport across cell membrane, and regulation of protein functions in membrane. The long term goal of the PI’s 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 this proposal is to test the hypothesis that oligomer stabilities of membrane proteins are optimized to support function and a significant increase of stability will decrease activity, using Escherichia coli protein AcrB as a model. A major difficulty in studying protein oligomers in cell membrane is a lack of techniques to characterize protein structure in the native state in the cell membrane, as most current structural characterization methods require the extraction of proteins from the membrane, which may affect the structure of proteins and cause oligomers to dissociate. In preliminary studies, the PI’s group has developed a set of novel tools that enabled the characterization of AcrB structure in the cell membrane under the native state. The hypothesis will be tested through pursuing the following research objectives: 1) To quantitatively determine the coupling between AcrB trimer stability and transport activity. In preliminary studies, the PI’s group has created more than 40 AcrB constructs with various levels of activities. All constructs contain mutations at the inter-subunit interface to disrupt trimer stability. The dependence of transport activity on the relative trimer stability of these mutants will be quantitatively determined. 2) To identify gain-of-function mutations that improve AcrB trimer stability. Mutations that restore functions of partially active mutants will be identified using a positive selection procedure. Mutations that restore function through improving trimer stability (gain-of-stability) will then be isolated. 3) To determine to what extent the introduction of gain-of-stability mutations affect the activity of fully functional AcrB. Gain-of-stability mutations will be introduced into the sequence of the wild type AcrB to create super stable trimers, and then the activity of the resultant AcrB constructs will be measured in vivo. Outcomes from these researches will reveal the influence of trimer stability on AcrB activity. It is anticipated that this study will generate some of the first data concerning the correlation between oligomer stability of multi-domain and multi-span helical membrane proteins and their in vivo transport activity. The protocols and parameters that will be developed in the proposed research will provide valuable tools and bench-marks for other investigators studying protein-protein interaction in the cell membrane and 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 high quality science education to multiple students of different background, including 2 graduate students and 2 to 3 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.
Effective start/end date5/1/124/30/17


Explore the research topics touched on by this project. These labels are generated based on the underlying awards/grants. Together they form a unique fingerprint.