Design and optimization of enzymatic activity in a de novo β-barrel scaffold.

While native scaffolds offer a large diversity of shapes and topologies for enzyme engineering, their often unpredictable behavior in response to sequence modification makes de novo generated scaffolds an exciting alternative. Here we explore the customization of the backbone and sequence of a de novo designed eight stranded β-barrel protein to create catalysts for a retro-aldolase model reaction. We show that active and specific catalysts can be designed in this fold and use directed evolution to further optimize activity and stereoselectivity.

Engineering Chemoselectivity in Hemoprotein-Catalyzed Indole Amidation.

Here we report a cytochrome P450 variant that catalyzes C-amidation of 1-methylindoles with tosyl azide via nitrene transfer. Before evolutionary optimization the enzyme exhibited two undesired side reactivities resulting in reduction of the putative iron-nitrenoid intermediate or cycloaddition between the two substrates to form triazole products. We speculated that triazole formation was a promiscuous cycloaddition activity of the P450 heme domain, while sulfonamide formation likely arose from surplus electron transfer from the reductase domain.

Design and biological testing of peptidic dimerization inhibitors of human Hsp90 that target the C-terminal domain.

Background: Small molecules targeting the dimerization interface of the C-terminal domain of Hsp90, a validated target for cancer treatment, have yet to be identified.

Methods: Three peptides were designed with the aim to inhibit the dimerization of Hsp90. Computational and biophysical methods examined the α-helical structure for the three peptides.