Sculpting Metal‐binding Environments in <i>De Novo</i> Designed Three‐helix Bundles

Plegaria, Jefferson S.; Pecoraro, Vincent L.

doi:10.1002/ijch.201400146

Cited by 15 publications

(15 citation statements)

References 71 publications

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“…After 14–18 h, the cells were harvested by centrifugation and lysed by a microfluidizer, while peptide was purified using previously published methods. 51 Final OD 600 ’s of these autoinduction cultures varied between 6 and 16, with purified peptide yields of 30–200 mg/L.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2018

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Cupredoxins are copper-dependent electron-transfer proteins that can be categorized as blue, purple, green, and red depending on the spectroscopic properties of the Cu(II) bound forms. Interestingly, despite significantly different first coordination spheres and nuclearity, all cupredoxins share a common Greek Key β-sheet fold. We have previously reported the design of a red copper protein within a completely distinct three-helical bundle protein, α3DChC2.1 While this design demonstrated that a β-barrel fold was not requisite to recapitulate the properties of a native cupredoxin center, the parent peptide α3D was not sufficiently stable to allow further study through additional mutations. Here we present the design of an elongated protein GRANDα3D (GRα3D) with ΔGu = −11.4 kcal/mol compared to the original design’s −5.1 kcal/mol. Diffraction quality crystals were grown of GRα3D (a first for an α3D peptide) and solved to a resolution of 1.34 Å. Examination of this structure suggested that Glu41 might interact with the Cu in our previously reported red copper protein. The previous bis(histidine)(cysteine) site (GRα3DChC2) was designed into this new scaffold and a series of variant constructs were made to explore this hypothesis. Mutation studies around Glu41 not only prove the proposed interaction, but also enabled tuning of the constructs’ hyperfine coupling constant from 160 to 127 × 10−4 cm−1. X-ray absorption spectroscopy analysis is consistent with these hyperfine coupling differences being the result of variant 4p mixing related to coordination geometry changes. These studies not only prove that an Glu41–Cu interaction leads to the α3DChC2 construct’s red copper protein like spectral properties, but also exemplify the exact control one can have in a de novo construct to tune the properties of an electron-transfer Cu site.

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Section: Methodsmentioning

confidence: 99%

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

et al. 2018

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“…Widening possibilities beyond low-molecular-weight complexes, rationally designed self-assembling peptidic scaffolds with well-defineds econdary and tertiary structuresa re tools of choice to mimic the structure and activity of an enzyme. [34][35][36][37][38][39][40][41][42] Only one manganese SOD mimic has been reported, which uses this type of construct with modest SOD activity (k cat = 3.7 10 5 m À1 s À1 at pH 7.4). [43] By using protein redesign Benson et al have obtained af unctional iron SOD mimic by introduc-ing aH is 3 metal-binding site and ap ocket for O 2 binding into E. coli thioredoxin (k cat = 6.410 6 m À1 s À1 at pH 8).…”

Section: Sodmentioning

confidence: 99%

Rational De Novo Design of a Cu Metalloenzyme for Superoxide Dismutation

Mathieu

Tolbert

Koebke

et al. 2019

Chemistry A European J

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Superoxide dismutases (SODs) are highly efficient enzymes for superoxide dismutation and the first line of defense against oxidative stress. These metalloproteins contain a redox‐active metal ion in their active site (Mn, Cu, Fe, Ni) with a tightly controlled reduction potential found in a close range around the optimal value of 0.36 V versus the normal hydrogen electrode (NHE). Rationally designed proteins with well‐defined three‐dimensional structures offer new opportunities for obtaining functional SOD mimics. Here, we explore four different copper‐binding scaffolds: H3 (His3), H4 (His4), H2DH (His3Asp with two His and one Asp in the same plane) and H3D (His3Asp with three His in the same plane) by using the scaffold of the de novo protein GRα3D. EPR and XAS analysis of the resulting copper complexes demonstrates that they are good CuII‐bound structural mimics of Cu‐only SODs. Furthermore, all the complexes exhibit SOD activity, though three orders of magnitude slower than the native enzyme, making them the first de novo copper SOD mimics.

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“…In spite of their relative simplicity, coiled coils are highly organised structures in which well defined 'active sites' can be engineered. [80][81][82][83][84][85][86] In addition to the lanthanides which will be covered in this review, examples of other metals that have been incorporated into coiled coils include, but are not limited to, first row transition metals such as zinc, 87 iron, 88 copper, 89 and heavy metals associated with toxicity such as mercury 90 and cadmium, 91 as well as systems incorporating multiple metal binding sites. [92][93][94][95] One advantage of coiled coil ligands is the capacity to control both the primary and secondary coordination spheres of bound metals by small changes in the amino acid sequence.…”

Section: De Novo Designed Peptide Coiled Coils As Ligandsmentioning

confidence: 99%