Structure-function Studies of the Allosteric Mechanisms of Protein Switches

开关蛋白的活性可以根据信号分子的结合来开启和关闭。这些效应是细胞如何执行复杂功能的核心。蛋白质工程师的动机是制造他们自己设计的开关，以测试他们对自然开关的理解，测试关于蛋白质形式和功能的分子基础的假设，创造用于阐明细胞功能和行为的工具，并创造用于传感和生物医学应用的开关。PI通过融合编码麦芽糖结合蛋白(MBP)和TEM1β-内酰胺酶(BLA)的基因来创造开关。了解这种开关的机制将为研究这种效应是如何通过进化而出现的提供信息，并将促进未来应用的开关构造。这项研究的目标是加深对开关蛋白的理解，并建立可用于构建开关的设计原则。MBP-BLA开关RG13的结构模型将结合核磁共振、计算建模和结晶学进行开发。这个模型将被用来提出关于转换机制的假设，然后将使用生物物理、生化和突变技术进行实验测试。广泛的影响核磁共振和计算建模工作将为研究大型蛋白质提供工具。一门结合计算蛋白质设计和定向进化方法的高级本科生/研究生课程将被共同教授，其中包括一个关于蛋白质开关工作的模块，部分原因是为了说明跨学科方法如何协同解决科学问题。这笔助学金资助的研究生将担任本课程的助教。接受培训的研究生将有来自不同科学领域的合作顾问，接受跨学科培训，并参加专业协会的会议。本科生和高中生将参与计划中的研究，增加三名教授在现有项目中的参与度，这些项目为一所以非裔美国人为主的高中的高中生提供研究经验。

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 ImpactsThe 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.