Grants and Contracts Details
Description
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.
Status | Finished |
---|---|
Effective start/end date | 5/1/12 → 4/30/17 |
Fingerprint
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.