CAREER: Biomolecular Engineering of Complex Protein Machinery in Living Cells

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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.