Confined enzyme catalysis in engineered protein nanoreactors

Nature generates a variety of nano-scale capsules. Most are composed of proteins. Viruses are the most well-known example, as they have a protein coat protecting the viral molecules. The proteins that form the coat, for example, can be assembled independently of the virus. The resulting nanocontainers can encapsulate DNA, RNA, enzymes, or drug molecules, for example. Nanoreactors contain enzymes and other catalysts and house specific reactions. A new class of bacterial proteins spontaneously form nano-vessels called encapsulins. This project will study the properties of encapsulins. Of particular interest is the ability to allow reactants and cofactors to enter the vessel and products to leave. This project also includes training opportunities for undergraduate students and postdoctoral fellows and supports undergraduate research in synthetic biology. A key problem exhibited by previously engineered nanoreactors is a mismatch between protein shell selectivity and the substrate and cofactor requirements of encapsulated enzymes. The overall objective of this project is to build a generalizable and modular nanoreactor platform based on pore-engineered encapsulin protein shells. Initially, encapsulin-based protein shells, optimized for small molecule flux, will be designed and characterized. Structure-guided rational design and pore engineering will be combined to create novel protein shell prototypes. The general goal is to increase the porosity of the protein shell without compromising the benefits of a protective physical barrier around an enzymatic cargo. The molecular flux across engineered protein shells will be tested and the activity of encapsulated glucokinase (GK) will be investigated and compared to unencapsulated GK. 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.