Protein Activity and Oligomer Stability in Cell Membrane

University Of Kentucky Research Foundation, Lexington KY

Investigators

Abstract

Intellectual Merit: The selective permeability of cell membranes, which is essential for all life forms that 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 membranes. However, questions remain about how proteins oligomerize in the cell membrane and how oligomerization determines protein functions. A thorough understanding of these questions will have an immense impact on many research areas including the biogenesis of cell membranes, establishment of homeostasis, signal transduction, material transport across cell membranes and regulation of protein functions in membranes. As a step toward a fundamental understanding of the preference for oligomer fomation, this project is designed to test the hypothesis that oligomer stabilities of membrane proteins are optimized to support function and a significant increase of stability will decrease activity. Escherichia coli protein AcrB will be used as a model. The hypothesis will be tested through creation and characterization of AcrB mutants with decreased or increased oligomer stability. An array of biochemical and biophysical methods will be used, including site-directed and random mutagenesis, protein extraction and purification, a protein activity assay, disulfide trapping, gel electrophoresis, circular dichroism and fluorescence spectroscopy. Outcomes from this research will reveal the influence of quaternary structure stability on AcrB function. This study is expected to generate some of the first data pertaining to the correlation between oligomer stability of multi-domain and multi-span helical membrane proteins and their in vivo activity. Broader Impacts: This work will involve a wide range of student training from high school to graduate candidates pursing Ph.D. degrees. Two graduate students will be trained to tackle scientific questions in the research area of membrane protein structure, function, folding, and interaction. In addition, two to three undergraduate and high school students will participate in the proposed research, through which they will gain hands-on experience in undertaking research in modern biochemistry. Special focus will be given to the recruitment and education of underrepresented and minority undergraduate students. The PI will provide an opportunity for students to work in a multidisciplinary and multicultural environment, preparing them for the increasingly international and global working environment. The discoveries resulting from the proposed research will be disseminated to scientific communities at large, through publications in peer-reviewed journals and presentations at research conferences.