Low resolution structure of the proteolytic fragments of the Rapana venosa hemocyanin in solution

Rapana venosa hemocyanin (Hc) is a giant oxygenbindingprotein consisting of different subunits assembledin a hollow cylinder. The polypeptide chain ofeach subunit is believed to be folded in several oxygenbindingfunctional units of molecular mass 50 kDa,each containing a binuclear copper active site. Limitedproteolysis with a-chymotrypsin of native R.venosa hemocyanin allows the separation of threefunctional proteolytic fragments with molecularmasses of '150, 100, and 50 kDa. The functional fragments,purified by combining gel filtration chromatographyand ion-exchange FPLC, were analyzed bymeans of small-angle X-ray scattering (SAXS). The gyrationradius of the 50-kDa Rapana Hc fraction (2.4nm) agrees well with that calculated on the basis of thedimensions determined by X-ray crystallography forone functional unit of Octopus Hc (2.1 nm). Independentshape determination of the 50- and 100-kDa proteolyticfragments yields consistent low-resolutionmodels. Simultaneous fitting of the SAXS data fromthese fragments provides a higher-resolution model ofthe 100-kDa species made of two functional units tiltedwith respect to each other. The model of the 150-kDaproteolytic fragment consistent with the SAXS datadisplays a linear chain-like aggregation of the 50-kDafunctional units. These observations provide valuableinformation for the reconstruction of the three-dimensionalstructure of the minimal functional subunit ofgastropod hemocyanin in solution. Furthermore, thespatial relationships among the different functionalunits within the subunit will help in elucidation of theoverall quaternary structure of the oligomeric nativeprotein.[...]