De Novo Design and Characterization of Copper Metallopeptides Inspired by Native Cupredoxins

Abstract

Using de novo protein design, we incorporated a copper metal binding site within the three-helix bundle α3D (Walsh et al. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 5486–5491) to assess whether a cupredoxin center within an α-helical domain could mimic the spectroscopic, structural, and redox features of native type-1 copper (CuT1) proteins. We aimed to determine whether a CuT1 center could be realized in a markedly different scaffold rather than the native β-barrel fold and whether the characteristic short Cu–S bond (2.1–2.2 Å) and positive reduction potentials could be decoupled from the spectroscopic properties (ε600 nm = 5000 M−1 cm−1) of such centers. We incorporated 2HisCys(Met) residues in three distinct α3D designs designated core (CR), chelate (CH), and chelate-core (ChC). XAS analysis revealed a coordination environment similar to reduced CuT1 proteins, producing Cu–S(Cys) bonds ranging from 2.16 to 2.23 Å and Cu–N(His) bond distances of 1.92–1.99 Å. However, Cu(II) binding to the CR and CH constructs resulted in tetragonal type-2 copper-like species, displaying an intense absorption band between 380 and 400 nm (>1500 M−1 cm−1) and A∥ values of (150–185) × 10−4 cm−4. The ChC construct, which possesses a metal-binding site deeper in its helical bundle, yielded a CuT1-like brown copper species, with two absorption bands at 401 (4429 M−1 cm−1) and 499 (2020 M−1 cm−1) nm and an A∥ value ~30 × 10−4 cm−4 greater than its native counterparts. Electrochemical studies demonstrated reduction potentials of +360 to +460 mV (vs NHE), which are within the observed range for azurin and plastocyanin. These observations showed that the designed metal binding sites lacked the necessary rigidity to enforce the appropriate structural constraints for a Cu(II) chromophore (EPR and UV–vis); however, the Cu(I) structural environment and the high positive potential of CuT1 centers were recapitulated within the α-helical bundle of α3D.

Clarifying the Copper Coordination Environment in a de Novo Designed Red Copper Protein

“…1 Instead, analysis of models based on our X-ray structure suggested that a dissymmetrically oriented copper ion was located within the hydrophobic core of GR α 3 DChC2 and that this allowed surface residues access to the metal. We have shown through extensive site-directed mutation studies that a previously unconsidered amino acid designed as part of a salt bridge (glutamate 41) is imperative for Cu(II)GR α 3 DChC2’s nitrosocyanin-like spectroscopy.…”

“…Comparison of GR α 3 D to GR α 3 DChC2 gives a destabilizing effect of ∼8 kcal/mol at pH 8.5 (Table 3) for incorporation of the ChC2 metal binding site, as compared to destabilizations of 3.7, 3.8, and 1.9 kcal/mol for the other three cupredoxin models reported in previous work. 1 This destabilization of ChC2 incorporation is related not only to a decrease in C m but also to the slope m , which is linked to unfolding cooperativity. This change is likely due to the disruptive effect of mutating two core hydrophobic residues to His residues into the relatively tight confines of the hydrophobic core.…”

“…26–31 Cupredoxins have been the target of many metalloprotein design efforts due to their uniquely constrained metal binding centers and spectral properties. 4,6,7,17,32 Approaches employing protein redesign using azurin as a scaffold, 20,28,33–35 de novo design within three or four helical bundles, 1 , 36–38 and artificial metalloproteins 39 have been explored. DeGrado et al designed a single polypeptide that formed an antiparallel 3-helix bundle called α 3 D, 40,41 and this scaffold has been modified to serve as a heavy metal binding protein, 42 capable of binding Cd(II) or iron in a tetrathiolate center.…”

“…We have previously reported the design of a red copper protein within a completely distinct three-helical bundle protein, α 3 DChC2. 1 While this design demonstrated that a β -barrel fold was not requisite to recapitulate the properties of a native cupredoxin center, the parent peptide α 3 D was not sufficiently stable to allow further study through additional mutations. Here we present the design of an elongated protein GRAND α 3 D (GR α 3 D) with ΔG u = −11.4 kcal/mol compared to the original design’s −5.1 kcal/mol.…”

Modular Artificial Cupredoxins

“…Attempts to model these active sites include metallopeptides with sequences that contain the loop of cupredoxins 16 and three-helix bundles 17 that assemble to form a Type 1 Cu binding site. In addition, synthetic Cu complexes have been prepared with sterically constrained ligands that have been useful in investigating the details of the Cu II –S(thiolate) interaction.…”