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

ABSTRACT: 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 ?-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.

SUBMITTER: Tegoni M

PROVIDER: S-EPMC3535669 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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

Tegoni Matteo M Yu Fangting F Bersellini Manuela M Penner-Hahn James E JE Pecoraro Vincent L VL

Proceedings of the National Academy of Sciences of the United States of America 20121210 52

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) ce ...[more]

PMID: 23236170

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Project description:ARCIMBOLDO solves the phase problem by combining the location of small model fragments using Phaser with density modification and autotracing using SHELXE. Mainly helical structures constitute favourable cases, which can be solved using polyalanine helical fragments as search models. Nevertheless, the solution of coiled-coil structures is often complicated by their anisotropic diffraction and apparent translational noncrystallographic symmetry. Long, straight helices have internal translational symmetry and their alignment in preferential directions gives rise to systematic overlap of Patterson vectors. This situation has to be differentiated from the translational symmetry relating different monomers. ARCIMBOLDO_LITE has been run on single workstations on a test pool of 150 coiled-coil structures with 15-635 amino acids per asymmetric unit and with diffraction data resolutions of between 0.9 and 3.0?Å. The results have been used to identify and address specific issues when solving this class of structures using ARCIMBOLDO. Features from Phaser v.2.7 onwards are essential to correct anisotropy and produce translation solutions that will pass the packing filters. As the resolution becomes worse than 2.3?Å, the helix direction may be reversed in the placed fragments. Differentiation between true solutions and pseudo-solutions, in which helix fragments were correctly positioned but in a reverse orientation, was found to be problematic at resolutions worse than 2.3?Å. Therefore, after every new fragment-placement round, complete or sparse combinations of helices in alternative directions are generated and evaluated. The final solution is once again probed by helix reversal, refinement and extension. To conclude, density modification and SHELXE autotracing incorporating helical constraints is also exploited to extend the resolution limit in the case of coiled coils and to enhance the identification of correct solutions. This study resulted in a specialized mode within ARCIMBOLDO for the solution of coiled-coil structures, which overrides the resolution limit and can be invoked from the command line (keyword coiled_coil) or ARCIMBOLDO_LITE task interface in CCP4i.

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

Publications

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

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