Catalytically Active De Novo Proteins From Designed Combinatorial Libraries

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，大多数工作都集中在逐个蛋白质的设计上。相比之下，这项研究项目的目标是大规模设计蛋白质：研究的主要目标是(I)设计大量可折叠成稳定结构的新蛋白质文库，(Ii)从这些文库中分离出一系列在生化分析中具有功能活性的从头蛋白质，并从这些蛋白质中分离出具有支持生物生长能力的从头蛋白质。这些目标将通过完成以下四个具体目标来实现：1)将设计和构建一个高质量的从头α-螺旋蛋白质文库。与以前报道的文库相比，该文库将很大(~10^9)，几乎没有错误序列，并以稳定的折叠结构为主。2)通过对几种不同的酶活性使用高通量化学和生化筛选，将从该文库中分离催化活性从头蛋白。3)通过用户指定的机制完成催化的从头蛋白将通过基于机制的抑制物的筛选和选择而被分离。4)将使用强大的遗传选择来分离能够提供生物学功能的能够支持活细胞生长的新蛋白。作为细胞代谢的生物成分，这将是生物分子设计和合成生物学的一个重要里程碑。生物技术：按照今天的做法，生物技术主要是基于生产用于制药、农业和工业应用的蛋白质。尽管生物技术被吹捧为未来进步的关键驱动力，但目前使用的蛋白质产品基于相对较旧的技术，涉及克隆和生产天然蛋白质。虽然在某些情况下，这些天然蛋白质会被修饰以增强它们在特定应用中的适用性，但目前的蛋白质产品仍然是很容易识别的天然序列的衍生品。与序列空间中可能的蛋白质的数量和多样性相比，为当前生物技术提供“原料”的天然蛋白质的数量相形见绌。这一研究项目将探索大量从头设计的蛋白质的功能潜力，从而为未来针对特定应用设计、选择和生产新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，并学会开发自己的独立项目。他们还将培养教授、培训和指导研究小组中年轻和经验较少的成员的能力。与行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。这项工作的结果将在科学文献中传播，并在国内和国际会议上发表演讲(由学生和教职员工)。作为化学系本科生研究的主任，PI对鼓励本科生研究特别感兴趣；通常有4至6名本科生参与研究(包括出版物的联合作者)。该实验室鼓励代表性不足的人群参与科学研究。目前在Pi实验室的7名研究生中，5名是女性，1名是非裔美国人。该研究小组还参与了社区外展教育。该实验室有邀请高中科学教师在夏季到实验室工作的历史。此外，在过去的四个夏天里，来自该地区的高中生在其中一名研究生的指导下在实验室进行研究。化学系有一个外展项目，访问当地的学校教授动手科学，并鼓励PI的学生参加这个项目。

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.