Abstract
For decades the respiratory protein keyhole limpet hemocyanin (KLH1) from the marine gastropod Megathura crenulata has been used widely as a potent immunostimulant, useful hapten carrier, and valuable agent in the treatment of bladder carcinoma. Although much information on the immunological properties of KLH1 is available, biochemical and structural data are still incomplete. Small-angle x-ray scattering revealed the existence of two conformations, an oxy state being slightly more compact than the deoxy state. Based on small-angle scattering curves, a newly developed Monte Carlo algorithm delivered a surface representation of proteins. The massive changes of the surfaces of reconstructed didecameric KLH1 molecules are explained as a twist of the two non-covalently associated decameric half-molecules. Upon oxygenation, the KLH1 molecule becomes longer and skinnier. This study provides the first real evidence how a molluscan hemocyanin changes conformation during an allosteric transition.
Highlights
Two different hemocyanin isoforms, KLH1 and KLH2, have been found in the hemolymph of the keyhole limpet M. crenulata with molecular masses of ϳ8 MDa
Electron microscopy revealed similar quaternary structures as hollow cylinders with a D5 symmetry. It is composed of two isologeously dimerized decamers consisting of 10 large subunits, which fold into 7– 8 copper-containing functional units (FUs) of ϳ50 kDa being heterogeneous in their sequences [15, 16]
The arrangement of the subunits and their 160 FUs within the decamers is still under discussion, a 15-Å resolution three-dimensional reconstruction of KLH1 didecamer has been obtained from low temperature transmission electron microscopy [11]
Summary
Sample Preparation—KLH1 was kindly provided by Dr J. The whole cylinder was created by applying D5 symmetry operations As it was shown previously [28], the small-angle scattering intensity of a molecule can be calculated effectively from a random sample of the excess density. The whole procedure was repeated 20 times/data set with different seeds of the random number generator, giving a total of 100 models for oxygenated and deoxygenated KLH1. To reduce the statistical fluctuations, all of the individual models were superimposed and averaged density distributions were calculated for oxygenated and deoxygenated KLH1 molecules. This procedure compares only the relative handedness of the two different enantimorphic classes. We would like to point out that a different definition for is used in crystallographic papers where the error level is not calculated from different models but is estimated from the variation within one density distribution or difference density distribution, respectively
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