Collaborative Research: Engineering Hyperstable Enzymes via Computationally Guided Protein Stapling

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Enzymes are proteins that facilitate reactions. At modest temperatures or in response to pH changes, enzymes will lose their three-dimensional structure which leads to a loss in their activity as catalysts. If enzymes were more stable, then they could be used more widely in industrial applications. This research project will investigate a new method for designing proteins in which chemical links will be made between certain parts of the enzyme, also called "molecular stapling." This method might increase the stability of proteins at elevated temperatures or in the presence of chemicals. The activity of the new enzyme with "staples" should be unchanged from the original enzyme. The results of this project could lead to enzymes that could be designed to manufacture pharmaceuticals or to produce chemicals in a more sustainable manner. The research project serves as a training ground for undergraduate and graduate students to perform cutting edge research, leading to a skilled STEM workforce. The researchers will host local high school students for a research experience on enzymes and proteins. They will also participate in outreach to area community colleges.The idea behind this research project is that enzymes can be stabilized by genetically encoded, redox-stable covalent crosslinks. A novel computational design method for protein stabilization via covalent stapling will be developed via iterative design-build-test cycles, using a carbene transferase as testbed. The scope of this methodology will be extended to different stapling strategies for maximizing both thermodynamic and kinetic stability of the target enzyme, leading to hyperstabilization. Computational methodology development will be guided by detailed investigations of the stapled enzyme variants via functional assays and complementary biophysical techniques. The generality of this approach will be demonstrated through its application for the rapid and efficient thermostabilization of different enzymes of biotechnological importance. It is envisioned that this method will be broadly applicable to enzymes and proteins, providing an effective, and potentially general strategy for protein hyperstabilization.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.