Low-resolution structure of the proteolytic fragments of the Rapana venosa hemocyanin 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 functional units of molecular mass 50 kDa, each containing a binuclear copper active site. Limited proteolysis with alpha-chymotrypsin of native R. venosa hemocyanin allows the separation of three functional proteolytic fragments with molecular masses of approximately 150, 100, and 50 kDa. The functional fragments, purified by combining gel filtration chromatography and ion-exchange FPLC, were analyzed by means of small-angle X-ray scattering (SAXS). The gyration radius of the 50-kDa Rapana Hc fraction (2.4 nm) agrees well with that calculated on the basis of the dimensions determined by X-ray crystallography for one functional unit of Octopus Hc (2.1 nm). Independent shape determination of the 50- and 100-kDa proteolytic fragments yields consistent low-resolution models. Simultaneous fitting of the SAXS data from these fragments provides a higher-resolution model of the 100-kDa species made of two functional units tilted with respect to each other. The model of the 150-kDa proteolytic fragment consistent with the SAXS data displays a linear chain-like aggregation of the 50-kDa functional units. These observations provide valuable information for the reconstruction of the three-dimensional structure of the minimal functional subunit of gastropod hemocyanin in solution. Furthermore, the spatial relationships among the different functional units within the subunit will help in elucidation of the overall quaternary structure of the oligomeric native protein.
Dainese
E
E
Institute of Biochemistry, University of Teramo, Teramo, Italy.
Svergun
D
D
Beltramini
M
M
Di Muro
P
P
Salvato
B
B
D000818:Animals; D006433:Hemocyanins / Q000737:chemistry* / Q000302:isolation & purification; D008958:Models, Molecular; D008970:Molecular Weight; D008974:Mollusca / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011487:Protein Conformation; D012542:Scattering, Radiation; D012996:Solutions
D010446:Peptide Fragments; D012996:Solutions; D006433:Hemocyanins