De Novo Design of Copper Metallopeptides Capable of Electron Transfer: From Design to Function.

Abstract

Biophysical characterization on de novo designed three-helical bundles is presented. The structure was determined to establish its physical integrity. Spectroscopic, electrochemical and photophysical studies were used to characterize designed redox-active copper sites.

α3D, a de novo designed peptide that preassembles into a three-helix bundle fold, was functionalized with a triscysteine site to produce α3DIV. α3DIV structure was solved using Nuclear Magnetic Resonance (NMR). α3DIV comprised 1067 NMR restraints and 138 dihedral angles. The backbone of the 20 lowest energy structures has a root mean square deviation from the mean structure of 0.79 (0.16) Å, demonstrating a well-defined structure. The asymmetric 2HisCys(Met) copper electron transfer site, which is encompassed in the β-barrel fold of cupredoxins, was incorporated in α3D to examine whether the function and physical properties of cupredoxins can be recapitulated in an unrelated fold. This generated three designs: core, chelate and chelate-core constructs. Cu(II) binding to the core and chelate constructs displayed intense absorption bands between 380-400 nm (~2000 M−1 cm−1); the chelate-core construct showed two intense absorption bands at 401 (4429 M−1 cm−1) and 499 (2020 M−1 cm−1). X-ray absorption spectroscopy analysis on the Cu(I) adducts recapitulated the reduced state of cupredoxin proteins, producing short Cu-S(Cys) bonds at 2.16 – 2.23 Å. Overall, these results showed that the designed cupredoxin sites cannot enforce the structural constraints necessary for the appropriate Cu(II) chromophore, however the Cu(I) environment was retained. Moreover, the redox activity of the designed constructs was tested using electrochemical and photophysical methods. Electrochemical studies showed reduction potentials of +362 – +462 mV (vs. NHE), which are in the range of cupredoxins. Photophysical work revealed intermolecular ET activity with ruthenium(III)trisbipyridine produced first-order and bimolecular rate constants of 105 s−1 and 108 s−1 M−1, respectively.

This work illustrates that the redox function of a native copper center in a β-barrel fold can be achieved in the α-helical framework of α3D. Further, the structure of α3DIV revealed a distorted triscysteine site, offering a model for proteins with thiol-rich ligands. Ultimately, this work provides a foundation for investigating long-range electron transfer reaction using de novo protein design.