<i>De Novo</i>-Designed Enzymes as Small-Molecule-Regulated Fluorescence Imaging Tags and Fluorescent Reporters

Enzyme-based tags attached to a protein-of-interest (POI) that react with a small molecule, rendering the conjugate fluorescent, are very useful for studying the POI in living cells. These tags are typically based on endogenous enzymes, so protein engineering is required to ensure that the small-molecule probe does not react with the endogenous enzyme in the cell of interest. Here we demonstrate that <i>de novo</i>-designed enzymes can be used as tags to attach to POIs. The inherent bioorthogonality of the <i>de novo</i>-designed enzyme–small-molecule probe reaction circumvents the need for protein engineering, since these enzyme activities are not present in living organisms. Herein, we transform a family of <i>de novo</i>-designed retroaldolases into variable-molecular-weight tags exhibiting fluorescence imaging, reporter, and electrophoresis applications that are regulated by tailored, reactive small-molecule fluorophores

Computational Design of Enone-Binding Proteins with Catalytic Activity for the Morita–Baylis–Hillman Reaction

The Morita–Baylis–Hillman reaction forms a carbon–carbon bond between the α-carbon of a conjugated carbonyl compound and a carbon electrophile. The reaction mechanism involves Michael addition of a nucleophile catalyst at the carbonyl β-carbon, followed by bond formation with the electrophile and catalyst disassociation to release the product. We used Rosetta to design 48 proteins containing active sites predicted to carry out this mechanism, of which two show catalytic activity by mass spectrometry (MS). Substrate labeling measured by MS and site-directed mutagenesis experiments show that the designed active-site residues are responsible for activity, although rate acceleration over background is modest. To characterize the designed proteins, we developed a fluorescence-based screen for intermediate formation in cell lysates, carried out microsecond molecular dynamics simulations, and solved X-ray crystal structures. These data indicate a partially formed active site and suggest several clear avenues for designing more active catalysts