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

ABSTRACT: 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 ?-sheet fold. We have previously reported the design of a red copper protein within a completely distinct three-helical bundle protein, ?3DChC2. (1) While this design demonstrated that a ?-barrel fold was not requisite to recapitulate the properties of a native cupredoxin center, the parent peptide ?3D was not sufficiently stable to allow further study through additional mutations. Here we present the design of an elongated protein GRAND?3D (GR?3D) with ? Gu = -11.4 kcal/mol compared to the original design's -5.1 kcal/mol. Diffraction quality crystals were grown of GR?3D (a first for an ?3D 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 (GR?3DChC2) 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 ?3DChC2 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.

SUBMITTER: Koebke KJ

PROVIDER: S-EPMC6183058 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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

Koebke Karl J KJ Ruckthong Leela L Meagher Jennifer L JL Mathieu Emilie E Harland Jill J Deb Aniruddha A Lehnert Nicolai N Policar Clotilde C Tard Cédric C Penner-Hahn James E JE Stuckey Jeanne A JA Pecoraro Vincent L VL

Inorganic chemistry 20180918 19

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, α<sub>3</sub>DChC2. (1) While this design demons ...[more]

PMID: 30226758

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Project description:UnlabelledTo survive environmental challenges, biological systems rely on proteins that were selected by evolution to function in particular cellular and conditional settings. With the advent of protein design and synthetic biology, it is now possible to construct novel proteins that are not biased by eons of selection in natural hosts. The availability of these sequences prompts us to ask whether natural biological organisms can use naïve-non-biological-proteins to enhance fitness in stressful environments. To address this question, we transformed a library of DNA sequences encoding ?1.5 × 10(6) binary patterned de novo proteins into E. coli, and selected for sequences that enable growth in concentrations of copper that would otherwise be toxic. Several novel sequences were discovered, and one of them, called Construct K (ConK), was studied in detail. Cells expressing ConK accumulate approximately 50% less copper than control cells. The function of ConK does not involve an oxidase, nor does it require two of the best characterized copper efflux systems. However, the ability of ConK to rescue cells from toxic concentrations of copper does require an active proton motive force. Further selections for growth in higher concentrations of copper led to the laboratory evolution of variants of ConK with enhanced levels of activity in vivo. These studies demonstrate that novel proteins, unbiased by evolutionary history in the natural world, can enhance the fitness of biological systems.SynopsisLiving systems evolve to adapt to potentially lethal environmental changes. This normally involves repurposing existing genetic information (i.e. sequences that were selected by billions of years of evolution). Here we show that a completely de novo protein, not derived from nature, can enable E. coli cells to grow in otherwise toxic concentrations of copper, demonstrating that living systems also have the capacity to incorporate and protopurpose entirely novel genetic information.

Dataset Information

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

Publications

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

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