Metal-ion binding and limited proteolysis of betabellin 15D, a designed beta-sandwich protein.
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.
Guy
P A
PA
Department of Analytical Chemistry, Glaxo Wellcome, Inc., Research Triangle Park, North Carolina 27709, USA.
Anderegg
R J
RJ
Lim
A
A
Saderholm
M J
MJ
Yan
Y
Y
Erickson
B W
BW
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
D002413:Cations, Divalent; D004220:Disulfides; D008670:Metals; D010446:Peptide Fragments; D011506:Proteins; D011994:Recombinant Proteins; C114709:betabellin 15; D010434:Pepsin A