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Crystallization and preliminary analysis of crystals of the 24-meric hemocyanin of the emperor scorpion (Pandinus imperator).

Jaenicke E, Pairet B, Hartmann H, Decker H - PLoS ONE (2012)

Bottom Line: Hemocyanins are giant oxygen transport proteins found in the hemolymph of several invertebrate phyla.Subunit interactions within the multisubunit hemocyanin complex lead to diverse allosteric effects such as the highest cooperativity for oxygen binding found in nature.A preliminary analysis of the hemocyanin structure reveals that emperor scorpion hemocyanin crystallizes in the same oxygenated conformation, which is also present in solution as previously shown by cryo-EM reconstruction and small angle x-ray scattering experiments.

View Article: PubMed Central - PubMed

Affiliation: Institut für Molekulare Biophysik, Johannes Gutenberg-Universität, Mainz, Germany. elmar.jaenicke@uni-mainz.de

ABSTRACT
Hemocyanins are giant oxygen transport proteins found in the hemolymph of several invertebrate phyla. They constitute giant multimeric molecules whose size range up to that of cell organelles such as ribosomes or even small viruses. Oxygen is reversibly bound by hemocyanins at binuclear copper centers. Subunit interactions within the multisubunit hemocyanin complex lead to diverse allosteric effects such as the highest cooperativity for oxygen binding found in nature. Crystal structures of a native hemocyanin oligomer larger than a hexameric substructure have not been published until now. We report for the first time growth and preliminary analysis of crystals of the 24-meric hemocyanin (M(W) = 1.8 MDa) of emperor scorpion (Pandinus imperator), which diffract to a resolution of 6.5 Å. The crystals are monoclinc with space group C 1 2 1 and cell dimensions a = 311.61 Å, b = 246.58 Å and c = 251.10 Å (α = 90.00°, β = 90.02°, γ = 90.00°). The asymmetric unit contains one molecule of the 24-meric hemocyanin and the solvent content of the crystals is 56%. A preliminary analysis of the hemocyanin structure reveals that emperor scorpion hemocyanin crystallizes in the same oxygenated conformation, which is also present in solution as previously shown by cryo-EM reconstruction and small angle x-ray scattering experiments.

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Emperor scorpion hemocyanin crystal.The crystals have the form of small bipyramids and grow to a maximum dimension of 500 µm within weeks after their appearance. The light blue color of the crystals indicated that hemocyanin molecules in the crystal are at least partially oxygenated. This crystal was used to measure the dataset.
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pone-0032548-g001: Emperor scorpion hemocyanin crystal.The crystals have the form of small bipyramids and grow to a maximum dimension of 500 µm within weeks after their appearance. The light blue color of the crystals indicated that hemocyanin molecules in the crystal are at least partially oxygenated. This crystal was used to measure the dataset.

Mentions: Crystals of the 24-meric hemocyanin of the emperor scorpion (Pandinus imperator) were grown by the hanging-drop vapor diffusion method. Small bipyramidal shaped single crystals appeared between three and ten weeks after setup of the experiment. The crystals grew to a maximum dimension of 500 µm within weeks after their appearance (Fig. 1). The light blue color of the crystals indicated that hemocyanin molecules in the crystal are at least partially oxygenated. A complete dataset of a crystal, which diffracted to a resolution of 6.5 Å, was recorded. The crystals are monoclinc with space group C 1 2 1 and cell dimensions a = 311.61 Å, b = 246.58 Å and c = 251.10 Å (α = 90.00°, β = 90.02°, γ = 90.00°). The content of the asymmetric unit was analyzed by molecular replacement. A first analysis suggested that the asymmetric unit contains one copy of the 24-meric hemocyanin with a Matthew's coefficient (Vm) of 2.79 Å3/Da and a solvent content of 56%. For molecular replacement search a pseudoatomic model of the 24-meric hemocyanin taking into account the different subunit types was calculated based on the electron density of a recent 6.8 Å cryo-EM reconstruction [33] (Fig. 2). In order to keep preexisting information about the quaternary structure to a minimum, a first search was conducted searching for four copies of a hexamer cut out from the 24-meric hemocyanin model. However, this search did not produce a meaningful result. Accordingly a second attempt was made by searching for two copies of 12-mer cut out from the 24-mer. This search successfully produced a 24-mer model having the same quaternary structure as the 24-mer from the cryo-EM reconstruction. Thus the molecular mass in the asymmetrical unit of emperor scorpion hemocyanin crystals is one of the largest in the PDB databank comparable to ribosomes, human DNA-dependent protein kinase and erythrocruorin [39], [40], [41].


Crystallization and preliminary analysis of crystals of the 24-meric hemocyanin of the emperor scorpion (Pandinus imperator).

Jaenicke E, Pairet B, Hartmann H, Decker H - PLoS ONE (2012)

Emperor scorpion hemocyanin crystal.The crystals have the form of small bipyramids and grow to a maximum dimension of 500 µm within weeks after their appearance. The light blue color of the crystals indicated that hemocyanin molecules in the crystal are at least partially oxygenated. This crystal was used to measure the dataset.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3293826&req=5

pone-0032548-g001: Emperor scorpion hemocyanin crystal.The crystals have the form of small bipyramids and grow to a maximum dimension of 500 µm within weeks after their appearance. The light blue color of the crystals indicated that hemocyanin molecules in the crystal are at least partially oxygenated. This crystal was used to measure the dataset.
Mentions: Crystals of the 24-meric hemocyanin of the emperor scorpion (Pandinus imperator) were grown by the hanging-drop vapor diffusion method. Small bipyramidal shaped single crystals appeared between three and ten weeks after setup of the experiment. The crystals grew to a maximum dimension of 500 µm within weeks after their appearance (Fig. 1). The light blue color of the crystals indicated that hemocyanin molecules in the crystal are at least partially oxygenated. A complete dataset of a crystal, which diffracted to a resolution of 6.5 Å, was recorded. The crystals are monoclinc with space group C 1 2 1 and cell dimensions a = 311.61 Å, b = 246.58 Å and c = 251.10 Å (α = 90.00°, β = 90.02°, γ = 90.00°). The content of the asymmetric unit was analyzed by molecular replacement. A first analysis suggested that the asymmetric unit contains one copy of the 24-meric hemocyanin with a Matthew's coefficient (Vm) of 2.79 Å3/Da and a solvent content of 56%. For molecular replacement search a pseudoatomic model of the 24-meric hemocyanin taking into account the different subunit types was calculated based on the electron density of a recent 6.8 Å cryo-EM reconstruction [33] (Fig. 2). In order to keep preexisting information about the quaternary structure to a minimum, a first search was conducted searching for four copies of a hexamer cut out from the 24-meric hemocyanin model. However, this search did not produce a meaningful result. Accordingly a second attempt was made by searching for two copies of 12-mer cut out from the 24-mer. This search successfully produced a 24-mer model having the same quaternary structure as the 24-mer from the cryo-EM reconstruction. Thus the molecular mass in the asymmetrical unit of emperor scorpion hemocyanin crystals is one of the largest in the PDB databank comparable to ribosomes, human DNA-dependent protein kinase and erythrocruorin [39], [40], [41].

Bottom Line: Hemocyanins are giant oxygen transport proteins found in the hemolymph of several invertebrate phyla.Subunit interactions within the multisubunit hemocyanin complex lead to diverse allosteric effects such as the highest cooperativity for oxygen binding found in nature.A preliminary analysis of the hemocyanin structure reveals that emperor scorpion hemocyanin crystallizes in the same oxygenated conformation, which is also present in solution as previously shown by cryo-EM reconstruction and small angle x-ray scattering experiments.

View Article: PubMed Central - PubMed

Affiliation: Institut für Molekulare Biophysik, Johannes Gutenberg-Universität, Mainz, Germany. elmar.jaenicke@uni-mainz.de

ABSTRACT
Hemocyanins are giant oxygen transport proteins found in the hemolymph of several invertebrate phyla. They constitute giant multimeric molecules whose size range up to that of cell organelles such as ribosomes or even small viruses. Oxygen is reversibly bound by hemocyanins at binuclear copper centers. Subunit interactions within the multisubunit hemocyanin complex lead to diverse allosteric effects such as the highest cooperativity for oxygen binding found in nature. Crystal structures of a native hemocyanin oligomer larger than a hexameric substructure have not been published until now. We report for the first time growth and preliminary analysis of crystals of the 24-meric hemocyanin (M(W) = 1.8 MDa) of emperor scorpion (Pandinus imperator), which diffract to a resolution of 6.5 Å. The crystals are monoclinc with space group C 1 2 1 and cell dimensions a = 311.61 Å, b = 246.58 Å and c = 251.10 Å (α = 90.00°, β = 90.02°, γ = 90.00°). The asymmetric unit contains one molecule of the 24-meric hemocyanin and the solvent content of the crystals is 56%. A preliminary analysis of the hemocyanin structure reveals that emperor scorpion hemocyanin crystallizes in the same oxygenated conformation, which is also present in solution as previously shown by cryo-EM reconstruction and small angle x-ray scattering experiments.

Show MeSH
Related in: MedlinePlus