Collaborative Research: Structures, Mechanism, and Functional Relevance of Filament Formation by Non-Cytoskeletal Enzymes

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This project will increase the understanding of a newly discovered mechanism of controlling enzyme activity, which involves the formation of linear self-assemblies (i.e. filaments). In particular, the reason for this behavior, and those situations where such behavior is advantageous, will be uncovered. Such understanding will aid in the understanding of enzyme function in normal biology, but also in many different diseases as well. The behavior can also be harnessed for new applications such as in biotechnology, material science, and the creation of new diagnostic tools. Broader impacts to society include opportunities for training of students at the high school, undergraduate, graduate, and postdoctoral levels in scientific approaches and methods. Outreach and recruiting efforts will be made to broaden opportunities for training to include members from communities under-represented in science. The overall scientific goal of this project is to discover the purposes and advantages of regulation of enzyme activity via homogeneous, linear polymerization (or filamentation). Specifically, the hypotheses that this mechanism is superior in rapid activation, and in controlling substrate specificity to secondary substrates under only particular reaction conditions, will be tested using the SgrAI enzyme system. In addition, hypotheses derived from structural studies will be tested, namely in how filamentation activates the enzyme for DNA cleavage, and how DNA sequences of the two types of substrates control enzyme activity by controlling filamentation. Finally, the research will be broadened to include a second filament forming enzyme, phosphofructokinase-1, to develop a full kinetic model so that key advantages of filamentation to this enzyme's regulation may be identified. Methodologies to be used include global kinetic modeling of enzyme activity data derived from single turnover reactions and detected via changes in FRET signals or conversion of radiolabeled substrates to products. Structural studies will utilize state-of-the-art cryo-electron microscopy and x-ray crystallographic methods. Mutagenesis will be used to create variants of the enzymes to test specific hypotheses, and DNA cleavage or fructose phosphorylation assays used to measure reactivity and substrate specificity.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.