A Systematic Approach to Engineer Fyn Kinase Biosensor

描述(申请人提供)：基因编码的荧光蛋白(FP)的发现和发展使活细胞和动物的分子定位可视化成为生物学和医学的革命性进展。一种开发和优化基于FRET的生物传感器的通用和高效的策略可以改变生物传感器的发展领域，并允许对不同的细胞内分子进行详细的研究，以了解和治疗人类疾病。Fyn激酶在调节T细胞受体信号转导、学习记忆、细胞黏附、机械转导和前列腺癌发生等多种重要生物学功能中发挥重要作用。因此，我们建议开发一种基于定向进化的系统策略来开发和优化Fyn FRET生物传感器，以利用高效FRET对来可视化活细胞中的亚细胞FYN活性。因此，我们提出了两个具体的目标：(1)优化Fyn激酶底物多肽序列；(2)开发优化的SH2结构域和Fyn FRET生物传感器。这种系统和定向的进化方法原则上可以很容易地扩展到任何基于FP的遗传编码的FRET生物传感器的开发中。所获得的信息也将促进我们对FYN调控的分子机制的深入理解。因此，通过整合分子工程和活细胞成像方面的尖端技术，该提案的成功应成为彻底改变FRET生物传感器发展的起点，并对整个生物学研究产生革命性影响。

DESCRIPTION (provided by applicant): The discovery and development of genetically-encoded fluorescent proteins (FPs) have revolutionized biology and medicine by allowing the visualization of molecular localization in live cells and animals. A general and high-efficiency strategy for developing and optimizing FRET-based biosensor can transform the field of biosensor development and allow the detailed investigation of different intracellular molecules, for the purpose of understanding and treating human diseases. Fyn kinase plays crucial roles in regulating a variety of important biological functions, including T cell receptor signaling, learnig and memory, as well as cell adhesion, mechanotransduction, and prostate cancer development. Therefore, we propose to develop a systematic strategy based on directed evolution to develop and optimize a Fyn FRET biosensor for the visualization of the subcellular Fyn activity in live cells, utilizing a high-efficiency FRET pair. Accordingly, two specific aims are proposed: (1) Optimize a substrate peptide sequence for Fyn kinase; (2) Develop an optimized SH2 domain and Fyn FRET biosensor. This systematic and directed evolution method can be readily extended for the development of, in principle, any FP-based genetically-encoded FRET biosensor. The information obtained should also advance our in-depth understanding of the molecular mechanism underlying the Fyn regulation. Hence, by integrating cutting-edge technologies in molecular engineering and live cell imaging, the success of the proposal should serve as a starting point to revolutionize the development of FRET biosensors and have transformative impact on biological studies in general.