RoL: EAGER: DESYN-C3: Programmable control of metabolism in synthetic cells using intrinsically disordered proteins

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Almost all proteins have a specific three-dimensional structure that maintains its specific activity in the cell. One class of proteins do not. They are referred to as intrinsically disordered proteins (IDPs). Their role in the cell appears to be to spontaneously associate with other proteins and activate them collectively. This is an important function to include in the design of a synthetic cell. This project will explore how IDPs interact with other proteins and with DNA and RNA. Of particular interest is how those interactions affect the regulation of gene expression and cell metabolism. Dysfunctional interactions are linked to neurodegenerative disorders, such as ALS (Lou Gehrig's disease). The results of this project may lead to enhanced treatments and improved outcomes for patients suffering from these diseases. The project will also support training and education of undergraduate and graduate students. Members of underrepresented groups will take part via science involvement programs. This project will develop interactions between IDPs and nucleic acids as a control mechanism for gene expression. The approach will be extended to the regulation of protein synthesis. To accomplish that, IDPs will be modified to bind and sequester mRNA in membrane-free organelles. The IDP-RNA complexes will be designed to be responsive to environmental cues, including protein concentration, temperature and protease activity. They must also bind the target RNA tightly, and release captured RNA for expression on demand. Testing of these concepts will take place in a cell-free protein synthesis system. Transcriptional networks will be augmented with genes that express engineered nucleic acid-binding proteins. Pathways will be created to regulate expression of a simple reporter protein and to build circuits to regulate CRISPR interference systems. Thus, this project will establish a global control mechanism for synthetic cells to adapt their functioning to available environmental resources and cues.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.