CAREER: Biomolecular Engineering of Complex Protein Machinery in Living Cells

职业：活细胞中复杂蛋白质机械的生物分子工程

• 批准号: 0449080
• 负责人: Matthew DeLisa
• 金额: $ 40万
• 依托单位: Cornell University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0449080&HistoricalAwards=false
• 关键词: CAREER; Biomolecular; Engineering; Complex; Protein

0449080DeLisaAbstractDirected evolution has become a widely used tool for protein and nucleic acid design. Both establishing structure-function relationships and creating improved enzyme catalysts or ligand-binding proteins are enabled. In the majority of published reports, the target of directed evolution experiments is typically a single protein. But while virtually all major enzyme functions have been successfully targeted, there is almost a complete lack of studies that target multi-protein cellular machinery. In this proposal, techniques for analyzing and engineering a model complex protein machine, namely the bacterial twin-arginine translocation (Tat) machinery, will be developed. The proposed studies will utilize a strategy termed directed co-evolution to: (i) elucidate how the Tat machinery first recognizes it substrates and how it is able to discriminate between folded and misfolded proteins; and (ii) "reprogram" the Tat machinery towards new functions. This research will shed light on a poorly understood biological mechanism and will lead to the harnessing of this machinery for both the expression of commercially important proteins and the identification of correctly folded protein sequences. The research plan will be closely integrated with an equally motivated education and outreach program focused on the enrichment of conventional courses and the mentoring of young scientists in the area of biomolecular engineering. In parallel, an extended outreach program will be developed for disadvantaged students from New York City. The aim is to expose students to laboratory advances and breaking classroom subject matter.

定向进化已成为蛋白质和核酸设计中广泛使用的工具。既可以建立结构功能关系，也可以创建改进的酶催化剂或配体结合蛋白。在大多数已发表的报告中，定向进化实验的目标通常是单一蛋白质。但是，尽管几乎所有主要的酶功能都已成功靶向，但几乎完全缺乏针对多蛋白细胞机制的研究。在本提案中，分析和设计模型复杂蛋白质机器的技术，即细菌双精氨酸易位（Tat）机器，将被开发。拟议的研究将利用一种称为定向共同进化的策略：(i)阐明Tat机制如何首先识别底物，以及它如何能够区分折叠和错误折叠的蛋白质；（ii）“重新编程”Tat机制以实现新功能。这项研究将揭示一个鲜为人知的生物学机制，并将导致利用这一机制来表达商业上重要的蛋白质和识别正确折叠的蛋白质序列。该研究计划将与同样积极的教育和推广计划紧密结合，重点是丰富传统课程和指导生物分子工程领域的年轻科学家。与此同时，将为来自纽约市的弱势学生制定一个扩展的外展计划。目的是让学生接触实验室的进展和打破课堂主题。