Abstract
Glossoscolex paulistus is a free-living earthworm encountered in south-east Brazil. Its oxygen transport requirements are undertaken by a giant extracellular haemoglobin, or erythrocruorin (HbGp), which has an approximate molecular mass of 3.6 MDa and, by analogy with its homologue from Lumbricus terrestris (HbLt), is believed to be composed of a total of 180 polypeptide chains. In the present work the full 3.6 MDa particle in its cyanomet state was purified and crystallized using sodium citrate or PEG8000 as precipitant. The crystals contain one-quarter of the full particle in the asymmetric unit of the I222 cell and have parameters of a = 270.8 Å, b = 320.3 Å and c = 332.4 Å. Diffraction data were collected to 3.15 Å using synchrotron radiation on beamline X29A at the Brookhaven National Laboratory and represent the highest resolution data described to date for similar erythrocruorins. The structure was solved by molecular replacement using a search model corresponding to one-twelfth of its homologue from HbLt. This revealed that HbGp belongs to the type I class of erythrocruorins and provided an interpretable initial electron density map in which many features including the haem groups and disulfide bonds could be identified.
Highlights
The annelid erythrocruorins are giant hexagonal bilayer haemoglobins with molecular masses within the megadalton range
The crystal structures of two types of such erythrocruorins have far been described, those from Lumbricus terrestris (HbLt) (Royer et al, 2006) and Arenicola marina (HbAc) (Royer et al, 2007). In both cases the architecture of the full particle of approximately 3.6 MDa is based on two hexagonal discs in which the most prominent substructure corresponds to one-twelfth of the particle and is composed of a dodecamer of globins together with three nonglobin chains called linkers
The many previously described studies on HbGp, together with the research currently under way regarding its oligomeric stability, make it an attractive system on which to work and in this paper we describe progress made towards its structure determination
Summary
Its oxygen transport requirements are undertaken by a giant extracellular haemoglobin, or erythrocruorin (HbGp), which has an approximate molecular mass of 3.6 MDa and, by analogy with its homologue from Lumbricus terrestris (HbLt), is believed to be composed of a total of 180 polypeptide chains. In the present work the full 3.6 MDa particle in its cyanomet state was purified and crystallized using sodium citrate or PEG8000 as precipitant. The structure was solved by molecular replacement using a search model corresponding to one-twelfth of its homologue from HbLt. The structure was solved by molecular replacement using a search model corresponding to one-twelfth of its homologue from HbLt This revealed that HbGp belongs to the type I class of erythrocruorins and provided an interpretable initial electron density map in which many features including the haem groups and disulfide bonds could be identified
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