Catalytically Active De Novo Proteins From Designed Combinatorial Libraries

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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.