title: Metal-ion binding and limited proteolysis of betabellin 15D, a designed beta-sandwich protein.
abstract: Betabellin 15D is a 64-residue, disulfide-bridged homodimer. When folded into a beta structure, the protein is predicted to have two clusters of three histidine residues, each cluster able to bind a divalent metal ion. When the protein was incubated with Cu2+, Zn2+, Co2+, or Mn2+, metal complexes of betabellin 15D were observed by electrospray-ionization mass spectrometry. The relative abundances of the ionic complexes suggested an order of affinities of Cu2+ > Zn2+ > Co2+ > Mn2+, consistent with solution-phase affinities for nitrogen- or sulfur-containing ligands. Limited proteolysis of betabellin 15D by immobilized pepsin, as measured by nanoelectrospray-ionization mass spectrometry, showed that the Phe12-Ser13 peptide bond of betabellin 15D was cleaved more slowly in the presence of Cu2+ than in its absence. Because Cu2+ has little or no effect on the catalytic rate of pepsin, the slower cleavage of the Phe12-Ser13 peptide bond may be due to its decreased accessibility caused by Cu(2+)-induced folding of betabellin 15D.
authors.affiliation: Department of Analytical Chemistry, Glaxo Wellcome, Inc., Research Triangle Park, North Carolina 27709, USA.
authors.forename: P A
authors.initials: PA
authors.lastname: Guy
authors.forename: R J
authors.initials: RJ
authors.lastname: Anderegg
authors.forename: A
authors.initials: A
authors.lastname: Lim
authors.forename: M J
authors.initials: MJ
authors.lastname: Saderholm
authors.forename: Y
authors.initials: Y
authors.lastname: Yan
authors.forename: B W
authors.initials: BW
authors.lastname: Erickson
mesh_terms: D000595:Amino Acid Sequence; D002413:Cations, Divalent / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D006868:Hydrolysis; D008670:Metals / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010434:Pepsin A; D010446:Peptide Fragments / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins
chemical_list: D002413:Cations, Divalent; D004220:Disulfides; D008670:Metals; D010446:Peptide Fragments; D011506:Proteins; D011994:Recombinant Proteins; C114709:betabellin 15; D010434:Pepsin A