CAREER: Engineering a Protein Molecular Switch

本研究的目的是开发和实施一种新的组合蛋白工程策略，称为随机结构域插入，将两种蛋白在功能上偶联以创建蛋白质分子开关。设计分子开关来调节或报告生物功能具有巨大的应用潜力，包括创建生物传感器、基因转录调节剂、代谢和细胞信号通路调节剂、药物递送伴侣、药物运输促进剂和新型生物材料。随机结构域插入策略的有效性将在两个模型中得到证明，“原理证明”系统使用特性良好的蛋白质。具有代表性的分子开关的动力学、结构和荧光行为将被表征，以便开发关于如何实现开关行为的模型。这些模型将进行实验检验。所开发的组合方法和模型将用于启动药物运输的分子开关工程。从中获得的科学见解将为各种应用的工程开关提供合理的基础，并为解决结构-功能关系问题提供独特的平台。在教育方面，PI将制定一个综合计划，为约翰霍普金斯大学的化学工程本科课程注入适当的组成部分，以培养对生物学有基本了解并能应用定量和非定量方法解决生物学问题的毕业生。这将包括生物工程实验课程的发展。

The objective of this proposed research is to develop and implement a novel, combinatorial protein engineering strategy called random domain insertion to couple two proteins functionally to create a protein molecular switch. The design of molecular switches to modulate or report on biological functions has enormous potential for a variety of applications including the creation of biosensors, regulators of gene transcription, modulators of metabolic and cell signaling pathways, chaperones for drug delivery, facilitators of drug transport, and novel biomaterials. The efficacy of the random domain insertion strategy will be demonstrated in two models, "proof-of-principle" systems using well-characterized proteins. The kinetic, structural, and fluorescent behavior of representative molecular switches will be characterized so as to develop models on how the switching behavior was achieved. These models will be tested experimentally. The combinatorial methods and models developed will be utilized to initiate engineering a molecular switch for drug transport. The scientific insights gained from this will provide a rational basis for engineering switches for a variety of applications as well as provide a unique platform for addressing questions of structure-function relationships. For the education component, the PI will be developing an integrated program for infusing the Chemical Engineering Undergraduate Program at Johns Hopkins University with the proper components to prepare graduates who have a basic understanding of biology and who can apply quantitative and non-quantitative approaches to biological problems. This will include the development of a Biological Engineering Laboratory course.