Analysis of the non-covalent interaction between metal ions and the cysteine-rich domain of protein kinase C eta by electrospray ionization mass spectrometry.
Effect of zinc and other metal ions on the folding of the protein kinase C (PKC) surrogate peptide (PKCeta-C1B) was analyzed intact under neutral conditions by electrospray ionization mass spectrometry (ESI-MS). ESI-MS spectrum of 64ZnCl(2)-folded PKCeta-C1B clearly showed that PKCeta-C1B coordinates specifically two atoms of zinc, and that the two thiol protons are lost in each zinc ion coordinate center. 113CdCl(2)-folded PKCeta-C1B also showed stoichiometry of two cadmium atoms that was proved by addition of EDTA. The dissociation constants of zinc- and cadmium-folded PKCeta-C1B in the phorbol 12,13-dibutyrate binding (PDBu) were similar (0.66 and 0.81 nM) with different B(max) values (46.4 and 71.4%). The difference would reflect higher coordination potency of cadmium ion that was demonstrated by ESI-MS when PKCeta-C1B was folded by 1:1 mixture of zinc and cadmium ions. In contrast, 63CuCl(2)-treated PKCeta-C1B did not show any copper-coordinated peak, instead a molecular mass less than 6 mass units smaller than that of apo-PKCeta-C1B was observed. The multiple charge mass envelope of copper-treated PKCeta-C1B shifted to that of the lower mass charge state like zinc-treated PKCeta-C1B. These data suggest that the copper treatment formed three intramolecular S-S bonds to abolish the PDBu binding of PKCeta-C1B.
Shindo
Mayumi
M
Applied Biosystems Japan Ltd., 4-5-4Hacchobori, Chuo-ku, Tokyo, 104-0032, Japan.
Irie
Kazuhiro
K
Fukuda
Hiroyuki
H
Ohigashi
Hajime
H
D003545:Cysteine / Q000378:metabolism*; D008670:Metals / Q000378:metabolism*; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D008670:Metals; D015240:Phorbol 12,13-Dibutyrate; C081975:protein kinase C eta; D011493:Protein Kinase C; D003545:Cysteine