Low-resolution structure of the proteolytic fragments of the Rapana venosahemocyanin in solution

Rapana venosa hemocyanin (Hc) is a giant oxygen-binding protein consisting of different subunits assembled in a hollow cylinder. The polypeptide chain of each subunit is believed to be folded in several oxygen-binding functio nal units of molecular mass 50 kDa, each containing a binuclear copper acti ve site. Limited proteolysis with alpha-chymotrypsin of native R. venosa he mocyanin allows the separation of three functional proteolytic fragments wi th molecular masses of approximate to 150, 100, and 50 kDa. The functional fragments, purified by combining gel filtration chromatography and ion-exch ange FPLC, were analyzed by means of small-angle X-ray scattering (SAXS). T he gyration radius of the 50-kDa Rapana He fraction (2.4 nm) agrees well wi th that calculated on the basis of the dimensions determined by X-ray cryst allography for one functional unit of Octopus He (2.1 nm). Independent shap e determination of the 50- and 100-kDa proteolytic fragments yields consist ent low-resolution models. Simultaneous fitting of the SAXS data from these fragments provides a higher-resolution model of the 100-kDa species made o f two functional units tilted with respect to each other. The model of the 150-kDa proteolytic fragment consistent with the SAXS data displays a linea r chain-like aggregation of the 50-kDa functional units. These observations provide valuable information for the reconstruction of the three-dimension al structure of the minimal functional subunit of gastropod hemocyanin in s olution. Furthermore, the spatial relationships among the different functio nal units within the subunit will help in elucidation of the overall quater nary structure of the oligomeric native protein. (C) 2000 Academic Press.