title: Purification and characterization of a novel calcium-binding protein from the extrapallial fluid of the mollusc, Mytilus edulis.
abstract: In the bivalve mollusc Mytilus edulis shell thickening occurs from the extrapallial (EP) fluid wherein secreted shell matrix macromolecules are thought to self-assemble into a framework that regulates the growth of CaCO(3) crystals, which eventually constitute approximately 95% of the mature shell. Herein is the initial report on the purification and characterization of a novel EP fluid glycoprotein, which is likely a building block of the shell-soluble organic matrix. This primary EP fluid protein comprises 56% of the total protein in the fluid and is shown to be a dimer of 28,340 Da monomers estimated to be 14.3% by weight carbohydrate. The protein is acidic (pI = 4.43) and rich in histidine content (11.14%) as well as in Asx and Glx residues (25.15% total). The N terminus exhibits an unusual repeat sequence of histidine and aspartate residues that occur in pairs: NPVDDHHDDHHDAPIVEHHD approximately. Ultracentrifugation and polyacrylamide gel electrophoresis demonstrate that the protein binds calcium and in so doing assembles into a series of higher order protomers, which appear to have extended structures. Circular dichroism shows that the protein-calcium binding/protomer formation is coupled to a significant rearrangement in the protein's secondary structure in which there is a major reduction in beta-sheet with an associated increase in alpha-helical content of the protein. A model for shell organic matrix self-assembly is proposed.
authors.affiliation: Departments of Chemistry and Biochemistry & Molecular Biology, University of New Hampshire, Durham, New Hampshire 03824, USA.
authors.forename: S J
authors.initials: SJ
authors.lastname: Hattan
authors.forename: T M
authors.initials: TM
authors.lastname: Laue
authors.forename: N D
authors.initials: ND
authors.lastname: Chasteen
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D049872:Bivalvia; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D014357:Trypsin / Q000737:chemistry
chemical_list: D000596:Amino Acids; D002135:Calcium-Binding Proteins; D014357:Trypsin