The activity of switch proteins can be turned on and off based on the binding of a signal molecule. Such effects are central to how cells carry out complex functions. Protein engineers are motivated to build switches of their own design to test their understanding of natural switches, to test hypotheses concerning the molecular basis of protein form and function, to create tools for elucidating cellular function and behavior, and to create switches for sensing and biomedical applications. The PI has created switches through fusing the genes encoding maltose binding protein (MBP) and TEM1 beta-lactamase (BLA). Understanding this switch's mechanism would inform the study of how such effects emerge through evolution and would facilitate future switch construction for applications. The objectives of this research effort are to develop an understanding of switch proteins as well as to establish design principles by which switches can be constructed. A structural model for MBP-BLA switch RG13 will be developed using a combination of NMR, computational modeling and crystallography. This model will be used to propose hypotheses on the switching mechanism and will then be tested experimentally using biophysical, biochemical and mutagenesis techniques.
Broader Impacts The NMR and computational modeling work will provide tools for the study of large proteins. A senior undergraduate/graduate course combining computational protein design and directed evolution approaches will be co-taught and include a module on the protein switch work, in part to illustrate how interdisciplinary approaches are synergistic for addressing scientific problems. Graduate students supported by this grant would serve as teaching assistants in this course. Graduate students trained would have co-advisors from different scientific fields, receive interdisciplinary training and participate in meetings of professional societies. Undergraduates and high school students will participate in the planned research, increasing the three professors' participation in existing programs that provide research experience to high school students from a high school with a predominantly African American student body.
This project provided new insights into the function and mechanism of a class of engineered proteins called "protein switches." These switches have promise for use as sensors, biomedical diagnostics, and selective therapeutic proteins. In addition, this study of engineered protein switches provided insight into the function of natural regulatory proteins. These insights came from gaining structural and biochemical insights into a model protein switch using spectroscopy (NMR) and protein mutagenesis. In addition, this study provided insight into the deleterious effects of mutation in the context of evolution. We found that computational predictions of protein stability were predictive of the ability of a bacterial protein to perform its function in bacterial cells. The entire study resulted in three papers describing our results, with one more currently in preparation. During the course of this research, three postdoctoral fellows, one undergraduate, and one high school student were trained in research. One postdoctoral fellow received training in molecular biology and protein NMR and presented her results at a national meeting. Two postdoctoral fellows received training in computational structure prediction and protein modeling. The undergraduate student and the high school student were trained in DNA mutagenesis, protein expression, protein purification, and enzymology. One postdoctoral fellow received experience in training and mentoring these two students.
