Arm Structure and Function in AraC Regulatory Protein

Johns Hopkins University, Baltimore MD

Investigators

Abstract

Intellectual Merit. AraC protein regulates expression of the L-arabinose operon, the genes that express proteins that breakdown the sugar arabinose as a source of energy in the bacterium Escherichia coli. The research will focus on the communication across the interface between the two structural domains of AraC, the arabinose-binding domain and the DNA-binding domain. This communication is necessary for the protein to respond to the presence of arabinose by changing its binding to DNA to regulate gene expression. In a series of steps of increasing resolution, the investigators will use computational protein docking, primarily using the Rosetta program suite, and laboratory experimentation, primarily using protein footprinting techniques, to precisely identify the inter-domain interfaces and interactions responsible for this communication. The N-terminal arm of AraC constitutes part of the inter-domain interface and its structural changes play a central role in governing the AraC response to arabinose. Molecular dynamics simulations with the CHARMM program will be used to determine the mechanistic basis by which mutations at residue 15 of the arm fail to shift AraC to the inducing state upon arabinose addition. Broader Impacts. The broader impacts of the research are as follows: Undergraduates and their professor, Mary Lowe, from a Loyola College, a local four year college, will participate on this work as well as undergraduate and graduate students from the Hopkins Biology, Biophysics, and Bioengineering Departments. Included in these will be members from under-represented minority groups that the Biology Department actively recruits to its graduate program and which the department's summer undergraduate research experiences (REU) program recruits from small colleges in the South. Additionally, the PI and two post graduate researchers will also participate in the research. In the third year of the project, the PI will write a manual to help students and research scientists use the powerful protein structure-prediction program Rosetta, similar to the textbook he has written on the use of the program CHARMM.