Title
Engineered Biomolecular Recognition of RDX by Using a Thermostable Alcohol Dehydrogenase as a Protein Scaffold
Author
Beyza Bulutoglu, Jennifer Haghpanah, Elliot Campbell, Scott Banta
Year
2018
Journal
ChemBioChem
Abstract
There are many biotechnology applications that would benefit from simple, stable proteins with engineered biomolecular recognition. Here, we explored the hypothesis that a thermostable alcohol dehydrogenase (AdhD from Pyrococcus furiosus) could be engineered to bind a small molecule instead of a cofactor or molecules involved in the catalytic transition state. We chose the explosive molecule 1,3,5-trinitro-1,3,5-triazine (royal demolition explosive, RDX) as a proof-of-concept. Its low solubility in water was exploited for immobilization for biopanning by using ribosome display. Docking simulations were used to identify two potential binding sites in AdhD, and a randomized library focused on tyrosine or serine mutations was used to determine that RDX was binding in the substrate binding pocket of the enzyme. A fully randomized binding pocket library was selected, and affinity maturation by error-prone PCR led to the identification of a mutant (EP-16) that gained the ability to bind RDX with an affinity of (73±11) μm. These results underscore the way in which thermostable enzymes can be useful scaffolds for expanding the biomolecular recognition toolbox.
Instrument
J-815
Keywords
Circular dichroism, Secondary structure, Thermal stability, Protein denaturation, Chemical stability, Biochemistry