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Abstract

Arthropod hemocyanins are large oligomeric oxygen-transporting proteins with molecular weight ranging from 450 kDa in the spiny lobster (Panulirus interruptus) up to more than 3.6 mDa in the horseshoe crab (Limulus polyphemus). Hemocyanins from different species consist of one or multiple copies of a hexameric building block (of 450 kDa) and are sufficiently large to be easily visualized in the electron microscope. Arthropod hemocyanins were among the first macromolecules studied by multivariate statistical image analysis techniques. We present an overview of the different characteristic molecular images of various multihexameric (1 × 6, 2 × 6, 4 × 6, and 8 × 6) assemblies as these occur in electron-microscopical preparations. We also model the different assemblies in three dimensions by merging multiple copies of the X-ray-diffraction electron density of the single hexameric hemocyanin of Panulirus interruptus. By making correct enantiomeric decisions while merging the densities at the various levels of assembly and by fine-tuning the assembly parameters used, a good match can be obtained between the microscopical images and two-dimensional projections calculated from the three-dimensional (3D) model densities. Knowledge of the quaternary structures of this intricate hierarchical family of oligomers is essential for understanding the allosteric interactions associated with their strong oxygen-binding cooperativity.