Catalytically Active De Novo Proteins From Designed Combinatorial Libraries

来自设计的组合文库的催化活性从头蛋白质

• 批准号: 0817651
• 负责人: Michael Hecht
• 金额: $ 48.67万
• 依托单位: Princeton University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2012-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0817651&HistoricalAwards=false
• 关键词: Catalytically; Active; De; Novo; Proteins

Intellectual Merit of the Research Project: A major challenge in Biomolecular Science is to construct novel macromolecules that fold into stable structures and catalyze chemical reactions. Significant progress in protein design has occurred in recent years; however, most of that work focuses on the design of individual proteins one-at-a-time. In contrast, this research project aims to design proteins on a large-scale: The key goals of the research are (i) to design vast libraries of novel proteins that fold into stable structures, and (ii) to isolate from these libraries a range of de novo proteins that are functionally active both in biochemical assays and in their abilities to support the growth of living organisms. These goals will be achieved by completing the following four specific aims: 1) A high quality library of de novo alpha-helical proteins will be designed and constructed. In contrast to libraries reported previously, this library will be large (~10^9), nearly free of error sequences, and dominated by stably folded structures.2) Catalytically active de novo proteins will be isolated from this library through the use of high throughput chemical and biochemical screens for several different enzymatic activities.3) De novo proteins that accomplish catalysis via a user specified mechanism will be isolated by subjecting the library to screens and selections based on mechanism-based inhibitors.4) Novel proteins that provide biological functions capable of supporting the growth of living cells will be isolated using powerful genetic selections.Broader Impacts Resulting from the Research Activity: Basic Science: Successful isolation of novel proteins that are active as chemical reagents in the test tube, and as biological components of cellular metabolism will represent a significant milestone in Biomolecular Design and Synthetic Biology. Biotechnology: As practiced today, biotechnology is based largely on the production of proteins for pharmaceutical, agricultural, and industrial applications. Although biotechnology is touted as a key driver for future advances, the protein products in use today are based on relatively old technologies involving the cloning and production of natural proteins. Although in some cases, these natural proteins are modified to enhance their suitability for particular applications, current protein products are nonetheless easily recognized derivatives of natural sequences. The number of natural proteins that provide the "feedstock" of current biotechnologies is dwarfed by the number and diversity of possible proteins in sequence space. This research project will explore the functional potential of large collections of proteins devised de novo, and thereby lay foundations for future technologies in which novel proteins are designed, selected, and produced for specific applications. Education and outreach: Students working on this research will enhance their experimental skills, and learn to develop their own independent projects. They will also develop an ability to teach, train, and mentor younger and less experienced members of the research group. Interactions with industrial collaborators will expose students to a variety of research options and employment opportunities. Results of the work will be disseminated in the scientific literature, and in presentations (by students & faculty) at national and international conferences.As the Chemistry Department's Director of Undergraduate Studies, the PI has a special interest in encouraging undergraduate research; 4 to 6 undergraduates are typically involved in the research (including co-authorship of publications.) The lab encourages the participation of underrepresented populations in scientific research. Of the 7 graduate students currently in PI's lab, 5 are women, and 1 is African American. The research group is also involved in community outreach education. The laboratory has a history of inviting high school science teachers to work in the lab during the summer. Moreover, for the past 4 summers, high school students from the area have done research in the lab under the mentorship of one of the graduate students. The Chemistry Department has an outreach program that visits local schools to teach hands-on science, and the PI's students are encouraged to participate in this program.

研究项目的智力优点：生物分子科学的主要挑战是建造新型的大分子，将其折叠成稳定的结构并催化化学反应。 近年来，蛋白质设计的显着进展发生了。但是，大多数工作都集中在一次性的单个蛋白质设计上。 相比之下，该研究项目旨在在大规模上设计蛋白质：研究的主要目标是设计大量的新型蛋白质图书馆，这些蛋白质的新型蛋白质折叠成稳定的结构，（ii）从这些图书馆中隔离一系列的DE从头蛋白质，这些蛋白质在生物化学测定中都具有功能性的活性，以在生物化学分析和能力中为其能力增长，以支持活着的活机体。 这些目标将通过完成以下四个特定目的来实现：1）将设计和构建从头α-螺旋蛋白的高质量图书馆。与之前报道的图书馆相反，该库将很大（〜10^9），几乎没有错误序列，并由稳定折叠的结构主导。2）催化活性的从该库中的催化活性从该库中分离出来，通过使用高吞吐量化学和生物化学筛选来实现几种不同的酶促效果，从而通过使用高吞吐量的蛋白质来隔离型蛋白质。3） 4）提供能够支持活细胞生长的生物学功能的新型蛋白质将使用强大的遗传选择来隔离。研究活动会产生研究活动：基础科学：基础科学的成功隔离，这些蛋白质的成功隔离，这些蛋白质是在测试管中作为化学反应的活性，以及​​作为生物学的生物学构成的生物学构成，代表了生物学的生物构成。 生物技术：正如今天实践的那样，生物技术主要基于用于制药，农业和工业应用的蛋白质的生产。 尽管生物技术被吹捧为未来进步的关键驱动力，但当今使用的蛋白质产品基于涉及自然蛋白的克隆和生产的相对旧技术。 尽管在某些情况下，这些天然蛋白会经过修改以增强其对特定应用的适用性，但是当前的蛋白质产品仍然是自然序列的衍生物。提供当前生物技术的“原料”的天然蛋白质数量与序列中可能蛋白的数量和多样性相形见war。该研究项目将探讨大量从头设计的蛋白质收集的功能潜力，从而为未来的技术奠定基础，在这些技术中，新型蛋白质被设计，选择和生产用于特定应用。 教育和宣传：从事这项研究的学生将提高他们的实验技能，并学会开发自己的独立项目。 他们还将发展研究小组的年轻且经验不足的成员的教学，培训和指导。 与工业合作者的互动将使学生了解各种研究选择和就业机会。这项工作的结果将在科学文献中传播，并在国家和国际会议上的演讲（由学生与教职员工）中传播。作为化学系的本科研究主任，PI对鼓励本科研究具有特别的兴趣； 4至6名本科生通常参与研究（包括出版物的共同著作。）该实验室鼓励人群不足的人群参与科学研究。 目前在Pi的实验室中的7名研究生中，有5名是女性，而1名是非裔美国人。 研究小组还参与了社区外展教育。实验室有邀请高中科学老师在夏季在实验室工作的历史。 此外，在过去的四个夏季中，该地区的高中生在一位研究生的指导下在实验室进行了研究。 化学系有一个外展计划，该计划访问当地学校教授动手科学，并鼓励PI的学生参加该计划。