2 research outputs found
<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
enzymeâsmall-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 MoritaâBaylisâHillman Reaction
The MoritaâBaylisâHillman reaction forms
a carbonâcarbon
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 ÎČ-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