A Systematic Approach to Engineer Fyn Kinase Biosensor

设计 Fyn 激酶生物传感器的系统方法

• 批准号: 8728967
• 负责人: Yingxiao Wang
• 金额: $ 18.79万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-21 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8728967
• 关键词: Affinity; Animals; Binding; Biological; Biological Assay; Biological Process; Biology; Biosensor; Cell Adhesion; Cell membrane; Cell physiology; Cells; Cellular biology; Development; Engineering; Event; Evolution; Family; Fluorescence; Fluorescence Resonance Energy Transfer; Focal Adhesions; Imagery; Immune response; In Vitro; Investigation; Learning; Libraries; Life; Lipids; Location; Malignant Neoplasms; Malignant neoplasm of prostate; Medicine; Membrane Microdomains; Memory; Methods; Molecular; Monitor; Monomeric GTP-Binding Proteins; Peptide Hydrolases; Peptides; Phosphorylation; Phosphotransferases; Play; Post-Translational Protein Processing; Process; Protein-Serine-Threonine Kinases; Proteins; Public Health; Receptor Signaling; Regulation; Reporting; Resistance; Resolution; Role; Sensitivity and Specificity; Signal Pathway; Signal Transduction; Signaling Molecule; Staging; Surface; T-Cell Receptor; Technology; Tyrosine; Yeasts; base; cellular imaging; directed evolution; flexibility; high throughput screening; human disease; improved; molecular domain; mutant; protein aminoacid sequence; screening; single-molecule FRET; spatiotemporal; src Homology Region 2 Domain; src-Family Kinases; success

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.

描述（由申请人提供）：遗传编码荧光蛋白（FP）的发现和开发通过允许活细胞和动物中分子定位的可视化而彻底改变了生物学和医学。开发和优化基于FRET的生物传感器的通用和高效策略可以改变生物传感器开发的领域，并允许详细研究不同的细胞内分子，以了解和治疗人类疾病。Fyn激酶在调节多种重要的生物学功能中起关键作用，包括T细胞受体信号传导、学习和记忆，以及细胞粘附、机械转导和前列腺癌的发展。因此，我们建议开发一个系统的策略，基于定向进化，开发和优化的Fyn FRET生物传感器的可视化的亚细胞Fyn活性在活细胞中，利用高效的FRET对。因此，本文提出了两个具体的目标：（1）优化Fyn激酶的底物肽序列;（2）开发优化的SH 2结构域和Fyn FRET生物传感器。这种系统和定向进化方法可以很容易地扩展，原则上，任何基于FP的基因编码的FRET生物传感器的发展。所获得的信息也应该推进我们深入了解Fyn调控的分子机制。因此，通过整合分子工程和活细胞成像的尖端技术，该提案的成功应该成为革命性FRET生物传感器发展的起点，并对一般生物学研究产生变革性影响。