De novo proteins provide a unique opportunity for investigating the structure-function relationships of metalloproteins in a minimal, well-defined, and controlled scaffold. Herein, we describe the rational programming of function in a de novo designed di-iron carboxylate protein from the due ferri family. Originally created to catalyze O2-dependent, two-electron oxidation of hydroquinones, the protein was reprogrammed to catalyze the selective N-hydroxylation of arylamines by remodeling the substrate access cavity and introducing a critical third His ligand to the metal binding cavity. Additional second-and third-shell modifications were required to stabilize the His ligand in the core of the protein. These changes resulted in at least a 106 –fold increase in the relative rates of the two reactions. This result highlights the potential for using de novo proteins as scaffolds for future investigations of geometric and electronic factors that influence the catalytic tuning of di-iron active sites.
The self-assembly of synthetic biomaterials, such as collagen peptides, can be harnessed for a range of biomedical applications. In an effort to obtain collagen-based macromolecular assemblies with temporal control, we designed a system that assembled only in the presence of external stimuli. We report a collagen triple helical peptide that is modified with a His(2) moiety on its C-terminus and a nitrilotriacetic acid unit on its N-terminus that rapidly and reversibly assembles in the presence of metal ions. Dynamic light scattering and turbidity experiments confirmed the presence of higher order aggregates in solution upon the introduction of Zn(2+), Cu(2+), Ni(2+), and Co(2+). This assembly process was found to be fully reversible using EDTA as a metal ion chelator. Control peptides that contain only a single ligand-modified terminus were not responsive to the same metal ions, thus demonstrating the requirement of both ligand modifications for peptide assembly. Scanning electron microscopy imaging of the peptide-metal assemblies revealed micrometer-sized florettes in addition to curved, stacked sheets. More detailed analysis of the Zn(2+)-generated microflorettes showed that the surface of these particles contains ruffled structures with a highly dense surface area. Potential folding intermediates in the formation of the microflorettes were observed at lower temperatures and at early time points in the assembly that are composed of curved layered sheets. Significantly, the assembly process proceeded under mild conditions using neutrally buffered aqueous solution at room temperature. These microscopic structures offer opportunities in many areas, including drug delivery, tissue engineering, and regenerative medicine.
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