De novo metalloprotein design is a remarkable approach to shape protein scaffolds toward specific functions. Here, we report the design and characterization of Due Rame 1 (DR1), a de novo designed protein housing a di‐copper site and mimicking the Type 3 (T3) copper‐containing polyphenol oxidases (PPOs). To achieve this goal, we hierarchically designed the first and the second di‐metal coordination spheres to engineer the di‐copper site into a simple four‐helix bundle scaffold. Spectroscopic, thermodynamic, and functional characterization revealed that DR1 recapitulates the T3 copper site, supporting different copper redox states, and being active in the O2‐dependent oxidation of catechols to o‐quinones. Careful design of the residues lining the substrate access site endows DR1 with substrate recognition, as revealed by Hammet analysis and computational studies on substituted catechols. This study represents a premier example in the construction of a functional T3 copper site into a designed four‐helix bundle protein.
De novo metalloprotein design is a remarkable approach to shape protein scaffolds toward specific functions. Here, we report the design and characterization of Due Rame 1 (DR1), a de novo designed protein housing a di‐copper site and mimicking the Type 3 (T3) copper‐containing polyphenol oxidases (PPOs). To achieve this goal, we hierarchically designed the first and the second di‐metal coordination spheres to engineer the di‐copper site into a simple four‐helix bundle scaffold. Spectroscopic, thermodynamic, and functional characterization revealed that DR1 recapitulates the T3 copper site, supporting different copper redox states, and being active in the O2‐dependent oxidation of catechols to o‐quinones. Careful design of the residues lining the substrate access site endows DR1 with substrate recognition, as revealed by Hammet analysis and computational studies on substituted catechols. This study represents a premier example in the construction of a functional T3 copper site into a designed four‐helix bundle protein.
Understanding the structural determinants for metal ion coordination in metalloproteins is a fundamental issue for designing metal binding sites with predetermined geometry and activity. In order to achieve this, we report in this paper the design, synthesis and metal binding properties of METP3, a homodimer made up of a small peptide, which self assembles in the presence of tetrahedrally coordinating metal ions. METP3 was obtained through a redesign approach, starting from the previously developed METP molecule. The undecapeptide sequence of METP, which dimerizes to house a Cys4 tetrahedral binding site, was redesigned in order to accommodate a Cys2His2 site. The binding properties of METP3 were determined toward different metal ions. Successful assembly of METP3 with Co(II), Zn(II) and Cd(II), in the expected 2:1 stoichiometry and tetrahedral geometry was proven by UV-visible spectroscopy. CD measurements on both the free and metal-bound forms revealed that the metal coordination drives the peptide chain to fold into a turned conformation. Finally, NMR data of the Zn(II)-METP3 complex, together with a retrostructural analysis of the Cys-X-X-His motif in metalloproteins, allowed us to define the model structure. All the results establish the suitability of the short METP sequence for accommodating tetrahedral metal binding sites, regardless of the first coordination ligands.
Designing metal sites into de novo proteins has significantly improved, recently. However, identifying the minimal coordination spheres, able to encompass the necessary information for metal binding and activity, still represents a great challenge, today. Here, we test our understanding with a benchmark, nevertheless difficult, case. We assemble into a miniature 28-residue protein, the quintessential elements required to fold properly around a FeCys4 redox center, and to function efficiently in electron-transfer. This study addresses a challenge in de novo protein design, as it reports the crystal structure of a designed tetra-thiolate metal-binding protein in sub-Å agreement with the intended design. This allows us to well correlate structure to spectroscopic and electrochemical properties. Given its high reduction potential compared to natural and designed FeCys4-containing proteins, we exploit it as terminal electron acceptor of a fully artificial chain triggered by visible light.
Designing metal sites into de novo proteins has significantly improved, recently. However, identifying the minimal coordination spheres, able to encompass the necessary information for metal binding and activity, still represents a big challenge, today. Here, we tested our understanding with a benchmark, nevertheless difficult, case. We assembled in a small 28-residue peptide the quintessential elements required to correctly fold around a single iron redox center, coordinated to four cysteinyl thiolates (FeCys4 site), and to efficiently function in electron-transfer. This study represents a milestone in de novo protein design: for the first time the crystal structure of a designed tetra-thiolate metal-binding protein is reported within sub-Å agreement with the intended design. This allowed us to well correlate structure to spectroscopic and electrochemical properties. Given its high reduction potential compared to natural and designed FeCys4-containing proteins, we exploited it as terminal electron acceptor of a fully artificial chain triggered by visible light.
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