The current trend in atmospheric carbon dioxide concentrations is causing increasing concerns for its environmental impacts, and spurring the developments of sustainable methods to reduce CO2 to usable molecules. We report the light-driven CO2 reduction in water in mild conditions by artificial protein catalysts based on cytochrome b562 and incorporating cobalt protoporphyrin IX as cofactor. Incorporation into the protein scaffolds enhances the intrinsic reactivity of the cobalt porphyrin toward proton reduction and CO generation. Mutations around the binding site modulate the activity of the enzyme, pointing to the possibility of further improving catalytic activity through rational design or directed evolution.
While covalent drug discovery is reemerging as an important
route
to small-molecule therapeutic leads, strategies for the discovery
and engineering of protein-based irreversible binding agents remain
limited. Here, we describe the use of yeast display in combination
with noncanonical amino acids (ncAAs) to identify irreversible variants
of single-domain antibodies (sdAbs), also called VHHs and nanobodies,
targeting botulinum neurotoxin light chain A (LC/A). Starting from
a series of previously described, structurally characterized sdAbs,
we evaluated the properties of antibodies substituted with reactive
ncAAs capable of forming covalent bonds with nearby groups after UV
irradiation (when using 4-azido-l-phenylalanine) or spontaneously
(when using O-(2-bromoethyl)-l-tyrosine).
Systematic evaluations in yeast display format of more than 40 ncAA-substituted
variants revealed numerous clones that retain binding function while
gaining either UV-mediated or spontaneous crosslinking capabilities.
Solution-based analyses indicate that ncAA-substituted clones exhibit
site-dependent target specificity and crosslinking capabilities uniquely
conferred by ncAAs. Interestingly, not all ncAA substitution sites
resulted in crosslinking events, and our data showed no apparent correlation
between detected crosslinking levels and distances between sdAbs and
LC/A residues. Our findings highlight the power of yeast display in
combination with genetic code expansion in the discovery of binding
agents that covalently engage their targets. This platform streamlines
the discovery and characterization of antibodies with therapeutically
relevant properties that cannot be accessed in the conventional genetic
code.
Protein-based self-assembled nanostructures hold tremendous promise as smart materials. One strategy to control the assembly of individual protein modules takes advantage of the directionality and high affinity bonding afforded by metal chelation. Here, we describe the use of 2,2'-bipyridine units (Bpy) as side chains to template the assembly of large structures (MW approx. 35 000 Da) in a metal-dependent manner. The structures are trimers of independently folded 3-helix bundles, and are held together by 2 Me(Bpy) complexes. The assemblies are stable to thermal denaturation, and are more than 90% helical at 90°C. Circular dichroism spectroscopy shows that one of the 2 possible (Bpy) enantiomers is favored over the other. Because of the sequence pliability of the starting peptides, these constructs could find use to organize functional groups at controlled positions within a supramolecular assembly.
While covalent drug discovery is reemerging as an important route to small molecule therapeutic leads, strategies for the discovery and engineering of protein-based irreversible binding agents remain limited. Here, we describe the use of yeast display, a high-throughput protein discovery platform, in combination with noncanonical amino acids (ncAAs) to identify irreversible variants of single-domain antibodies (sdAbs), also called VHHs and nanobodies, targeting botulinum neurotoxin light chain A (LC/A). Starting from a series of previously described, structurally characterized sdAbs, we evaluated the properties of antibodies substituted with reactive ncAAs capable of forming covalent bonds with nearby groups after UV irradiation (when using 4-azido-L-phenylalanine) or spontaneously (when using O-(2-bromoethyl)-L-tyrosine). Systematic evaluations in yeast display format of more than 40 ncAA-substituted variants revealed numerous clones that retain binding function while gaining either UV-mediated or spontaneous crosslinking capabilities. Solution-based analyses indicate that ncAA-substituted clones exhibit site-dependent target specificity and crosslinking capabilities uniquely conferred by ncAAs. Interestingly, not all ncAA substitution sites resulted in crosslinking events, and our data showed no apparent correlation between detected crosslinking levels and distances between sdAbs and LC/A residues. This underscores the utility of high-throughput platforms both to identify crosslinkable antibodies and to inform future rational and computational designs of such antibodies. Our findings highlight the power of yeast display in combination with genetic code expansion in the discovery of binding agents that covalently engage their targets. This platform streamlines the discovery and characterization of antibodies with therapeutically relevant properties that cannot be accessed in the conventional genetic code.
In nature, the majority of processes that occur in the cell involve the cycling of electrons and protons, changing the reduction and oxidation state of substrates to alter their chemical reactivity and usefulness in vivo. One of the most relevant examples of these processes is the electron transport chain, a series of oxidoreductase proteins that shuttle electrons through well-defined pathways, concurrently moving protons across the cell membrane. Inspired by these processes, researchers have sought to develop materials to mimic natural systems for a number of applications, including fuel production. The most common cofactors found in proteins to carry out electron transfer are iron sulfur clusters and porphyrin-like molecules. Both types have been studied within natural proteins, such as in photosynthetic machinery or soluble electron carriers; in parallel, an extensive literature has developed over recent years attempting to model and study these cofactors within peptide-based materials. This chapter will focus on major designs that have significantly advanced the field.
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