De novo protein design is an effective approach used to model the metal centers of native metalloproteins, which are active in fundamental biological reactions including catalysis and electron transfer. This approach involves the design and synthesis of a peptide scaffold from scratch, thus allowing one to incorporate an active site and achieve the function of a desired metal center in a simplified protein scaffold. We have incorporated a His2CysMet copper-binding motif within an antiparallel three-helix bundle to create a metal center that functions as an electron transfer site like that in the �-barrel fold of native cupredoxins. Circular dichroism studies show that the apo form of our designed cupredoxin construct is relatively well-folded and stable in solution. Preliminary, extended x-ray absorption fine structure analysis of a Cu(I) adduct reveals a coordination environment similar to that in reduced cupredoxins, with a short Cu-S and Cu-N bond distances of 2.22 � and 2.05 �, respectively. Cu(II) binding results in yellow species with an intense absorption band between 380-400 nm (~3000 M-1cm-1) instead of the blue (e600 nm = 6000 M-1 cm-1) or green (e450 nm = 1000 M-1 cm-1; e600 nm = 4000 M-1 cm-1) species reported for cupredoxins. Even though this spectroscopic feature deviates from its native counterpart, the redox properties of our copper-bound constructs demonstrate its ability to engage in electron transfer. From surface electrochemistry, we obtained a positive reduction potential between 380-400 mV (vs. NHE). Furthermore, photophysical studies by a flash-quench technique indicated an electron transfer reaction that involves the oxidation of the Cu(I) adduct by ruthenium(III)trisbipyridine.

Utilizing de novo protein design to create redox active copper peptides

248th National Meeting (2014)

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