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.
The objective of this project was to increase our understanding of the fundamental principles governing the action of proteins that regulate the expression on genes. In this project, the regulatory protein under study controls the expression of genes in the bacterium Escherichia coli. The action of the protein, AraC, was studied by two basic approaches, computational and experimental. In the computational approach, two classes of mutation were studied. Their behavior seemed paradoxical, and was not apparent from examination of the known structure of the protein. In both cases, molecular dynamics simulations revealed that the mutations adversely affected the structure of small clusters of hydrophobic amino acids, and the disruption of these clusters then prevented proper functioning of AraC. In the experimental approach, genetic studies hinted that the structure of a stretch of eight amino acids connecting two domains of the protein was of unexpected importance. Extensive genetic analysis confirmed the suspicion. These suggested examination of the flexibility of this linker region. A method of doing this based on fluorescence anisotropy was developed and indeed showed that binding of the effector of AraC, the sugar arabinose, leads to alteration of the linker flexibility. Future studies will be directed at determining how this flexibility is altered. Also aimed at determining details of the mechanism of arabinose response of AraC was the effort to determine the disposition of the two domains of AraC with respect to each other in the presence and absence of arabinose. Initial exploratory attempts revealed that the most informative approach would be to use the highly reactive hydroxy radical to modify the sidechains of surface-exposed residues on the domains. Comparison of the surface-exposed residues of the isolated individual domains with the residues exposed in the full protein in the presence and absence of arabinose reveals the protected, and hence contact regions between the domains. Identification of the surface-exposed residues is done using mass spectrometry to identify and quantitate the modified residues. Procedures for appropriate modification and their identification my mass spectrometry have been developed and future work will apply them to the final determination of the contact regions between the domains. Much of the computational work of this project was performed in collaboration with Professor Mary Lowe of Loyola University, a nearby undergradue institution. This work has produced a manuscript that is in the final stages of acceptance in a leading journal on protein structure and function. Dr. Lowe conducted her work with the assistance of a number of undergraduates from Loyola. In the summer and during vacation periods, the Loyola contingent attended many of our weekly group meetings. This research project contributed to the training of three undergraduates doing research in my laborator, as well as the training of three graduate students in my laboratory and many additional graduate students who rotated through my laboratory. It also enriched the graduate course in molecular biology which I teach to incoming graduate students and to my extensive web site, http://gene.bio.jhu.edu/, on our work and on related scientific issues,