Cu(I) binding properties of a designed metalloprotein

Xie, Fang; Sutherland, Duncan E. K.; Stillman, Martin J.; Ogawa, Michael Y.

doi:10.1016/j.jinorgbio.2009.12.005

Cited by 19 publications

(14 citation statements)

References 29 publications

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“…3b) equivalents respectively have been added per monomer. Cu(I)-thiolate clusters, such as those found in MTs, typically give rise to luminescence in the 500 to 700 nm region1214 due to solvent protected Cu(I)-Cu(I) interactions12495051. Such luminescence is observed and increases until ~9 to 11 (Supplementary Fig.…”

Section: Resultsmentioning

confidence: 98%

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Bacterial cytosolic proteins with a high capacity for Cu(I) that protect against copper toxicity

Vita

Landolfi

Baslé

et al. 2016

Sci Rep

199

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Bacteria are thought to avoid using the essential metal ion copper in their cytosol due to its toxicity. Herein we characterize Csp3, the cytosolic member of a new family of bacterial copper storage proteins from Methylosinus trichosporium OB3b and Bacillus subtilis. These tetrameric proteins possess a large number of Cys residues that point into the cores of their four-helix bundle monomers. The Csp3 tetramers can bind a maximum of approximately 80 Cu(I) ions, mainly via thiolate groups, with average affinities in the (1–2) × 1017 M−1 range. Cu(I) removal from these Csp3s by higher affinity potential physiological partners and small-molecule ligands is very slow, which is unexpected for a metal-storage protein. In vivo data demonstrate that Csp3s prevent toxicity caused by the presence of excess copper. Furthermore, bacteria expressing Csp3 accumulate copper and are able to safely maintain large quantities of this metal ion in their cytosol. This suggests a requirement for storing copper in this compartment of Csp3-producing bacteria.

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Section: Resultsmentioning

confidence: 98%

“…S7) equivalents of Cu(I) per monomer are added to Mt Csp3 (n = 8) and Bs Csp3 (n = 3) respectively. Emission then decreases to almost zero upon addition of further Cu(I), either due to structural modification of the original sites or an increase in their solvent exposure495051.…”

Section: Resultsmentioning

confidence: 99%

Bacterial cytosolic proteins with a high capacity for Cu(I) that protect against copper toxicity

Vita

Landolfi

Baslé

et al. 2016

Sci Rep

199

View full text Add to dashboard Cite

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“…Examples of Cu(I) (13,14) and Cu(II) de novo designed helix bundles have been reported (15)(16)(17)(18)(19)(20)(21)(22)(23)(24). However, only a few examples of controlled binding of copper at (His) 3 sites are known (17,20,21).…”

Section: +mentioning

confidence: 99%

Designing a functional type 2 copper center that has nitrite reductase activity within α-helical coiled coils

Tegoni

Yu²,

Bersellini

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

101

188

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One of the ultimate objectives of de novo protein design is to realize systems capable of catalyzing redox reactions on substrates. This goal is challenging as redox-active proteins require design considerations for both the reduced and oxidized states of the protein. In this paper, we describe the spectroscopic characterization and catalytic activity of a de novo designed metallopeptide Cu(I/II)(TRIL23H) 3 +/2+ , where Cu(I/II) is embeded in α-helical coiled coils, as a model for the Cu T2 center of copper nitrite reductase. In Cu(I/II)(TRIL23H) 3 +/2+ , Cu(I) is coordinated to three histidines, as indicated by X-ray absorption data, and Cu(II) to three histidines and one or two water molecules. Both ions are bound in the interior of the three-stranded coiled coils with affinities that range from nano- to micromolar [Cu(II)], and picomolar [Cu(I)]. The Cu(His) 3 active site is characterized in both oxidation states, revealing similarities to the Cu T2 site in the natural enzyme. The species Cu(II)(TRIL23H) 3 2+ in aqueous solution can be reduced to Cu(I)(TRIL23H) 3 + using ascorbate, and reoxidized by nitrite with production of nitric oxide. At pH 5.8, with an excess of both the reductant (ascorbate) and the substrate (nitrite), the copper peptide Cu(II)(TRIL23H) 3 2+ acts as a catalyst for the reduction of nitrite with at least five turnovers and no loss of catalytic efficiency after 3.7 h. The catalytic activity, which is first order in the concentration of the peptide, also shows a pH dependence that is described and discussed.

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“…[36] Interestingly, the inorganic cofactors were not only able to induce peptide self-assembly, but also to dictate the nature of the oligomerization state. [37,38] …”

Section: Introductionmentioning

confidence: 99%

Artificial Diiron Enzymes with a De Novo Designed Four‐Helix Bundle Structure

et al. 2015

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A single polypeptide chain may provide an astronomical number of conformers. Nature selected only a trivial number of them through evolution, composing an alphabet of scaffolds, that can afford the complete set of chemical reactions needed to support life. These structural templates are so stable that they allow several mutations without disruption of the global folding, even having the ability to bind several exogenous cofactors. With this perspective, metal cofactors play a crucial role in the regulation and catalysis of several processes. Nature is able to modulate the chemistry of metals, adopting only a few ligands and slightly different geometries. Several scaffolds and metal-binding motifs are representing the focus of intense interest in the literature. This review discusses the widespread four-helix bundle fold, adopted as a scaffold for metal binding sites in the context of de novo protein design to obtain basic biochemical components for biosensing or catalysis. In particular, we describe the rational refinement of structure/function in diiron–oxo protein models from the due ferri (DF) family. The DF proteins were developed by us through an iterative process of design and rigorous characterization, which has allowed a shift from structural to functional models. The examples reported herein demonstrate the importance of the synergic application of de novo design methods as well as spectroscopic and structural characterization to optimize the catalytic performance of artificial enzymes.

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Cu(I) binding properties of a designed metalloprotein

Cited by 19 publications

References 29 publications

Bacterial cytosolic proteins with a high capacity for Cu(I) that protect against copper toxicity

Bacterial cytosolic proteins with a high capacity for Cu(I) that protect against copper toxicity

Designing a functional type 2 copper center that has nitrite reductase activity within α-helical coiled coils

Artificial Diiron Enzymes with a De Novo Designed Four‐Helix Bundle Structure

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