CAREER: Biomolecular Engineering of Complex Protein Machinery in Living Cells

定向进化已经成为蛋白质和核酸设计中广泛使用的工具。 能够建立结构-功能关系和产生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质细胞机制的研究。 在这项建议中，将开发用于分析和工程化模型复杂蛋白质机器的技术，即细菌双精氨酸易位（达特）机器。 拟议的研究将利用一种称为定向共同进化的策略：（i）阐明达特机制如何首先识别它的底物，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”达特机制，使其具有新的功能。 这项研究将揭示一个知之甚少的生物学机制，并将导致利用这种机制来表达商业上重要的蛋白质和鉴定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，还将为纽约市处境不利的学生制定一个扩大的外联方案。 目的是让学生接触到实验室的进步和打破课堂主题。

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.