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.

该研究项目的智力价值：生物分子科学的一个主要挑战是构建能够折叠成稳定结构并催化化学反应的新型大分子。 近年来，蛋白质设计取得了重大进展；然而，大部分工作都集中在一次设计一个单独的蛋白质。 相比之下，该研究项目旨在大规模设计蛋白质：该研究的主要目标是（i）设计可折叠成稳定结构的庞大新型蛋白质库，以及（ii）从这些库中分离出一系列从头蛋白质，其在生化测定中和支持活生物体生长的能力方面均具有功能活性。 这些目标将通过完成以下四个具体目标来实现：1）将设计和构建高质量的从头α螺旋蛋白库。与之前报道的文库相比，该文库将很大（~10^9），几乎没有错误序列，并且以稳定折叠结构为主。2）通过使用高浓度从该文库中分离出具有催化活性的从头蛋白质。对几种不同的酶活性进行化学和生化筛选。3) 通过用户指定的机制完成催化作用的从头蛋白质将通过基于机制的抑制剂对文库进行筛选和选择来分离。4) 提供生物功能的新型蛋白质将利用强大的遗传选择来分离能够支持活细胞生长的蛋白质。研究活动产生的更广泛影响：基础科学：成功分离出在试管中作为化学试剂具有活性以及作为细胞代谢的生物成分的新型蛋白质将代表生物分子设计和合成生物学的一个重要里程碑。 生物技术：按照当今的实践，生物技术主要基于用于制药、农业和工业应用的蛋白质生产。 尽管生物技术被认为是未来进步的关键驱动力，但目前使用的蛋白质产品是基于相对较旧的技术，涉及天然蛋白质的克隆和生产。 尽管在某些情况下，这些天然蛋白质经过修饰以增强其对特定应用的适用性，但目前的蛋白质产品仍然是易于识别的天然序列的衍生物。与序列空间中可能的蛋白质的数量和多样性相比，提供当前生物技术“原料”的天然蛋白质的数量相形见绌。该研究项目将探索从头设计的大量蛋白质的功能潜力，从而为未来为特定应用设计、选择和生产新型蛋白质的技术奠定基础。 教育和推广：从事这项研究的学生将提高他们的实验技能，并学习开发自己的独立项目。 他们还将培养教学、培训和指导研究小组中年轻且经验不足的成员的能力。 与工业合作者的互动将使学生接触到各种研究选择和就业机会。工作成果将在科学文献中以及在国内和国际会议上（学生和教师）的演讲中传播。作为化学系本科生研究主任，PI 对鼓励本科生研究特别感兴趣；通常有 4 到 6 名本科生参与研究（包括共同撰写出版物）。实验室鼓励代表性不足的人群参与科学研究。 目前 PI 实验室的 7 名研究生中，5 名是女性，1 名是非裔美国人。 该研究小组还参与社区外展教育。该实验室有邀请高中科学教师暑期到实验室工作的历史。 此外，在过去的四个夏天，该地区的高中生在一名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，访问当地学校教授实践科学，鼓励 PI 的学生参加该计划。