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Binding studies of nNOS‐active amphibian peptides and Ca 2+ calmodulin, using negative ion electrospray ionisation mass spectrometry
Author(s) -
Pukala Tara L.,
Urathamakul Thitima,
Watt Stephen J.,
Beck Jennifer L.,
Jackway Rebecca J.,
Bowie John H.
Publication year - 2008
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.3757
Subject(s) - chemistry , calmodulin , mass spectrometry , peptide , electrospray ionization , stoichiometry , crystallography , calcium , chromatography , biochemistry , organic chemistry
Amphibian peptides which inhibit the formation of nitric oxide by neuronal nitric oxide synthase (nNOS) do so by binding to the protein cofactor, Ca 2+ calmodulin (Ca 2+ CaM). Complex formation between active peptides and Ca 2+ CaM has been demonstrated by negative ion electrospray ionisation mass spectrometry using an aqueous ammonium acetate buffer system. In all cases studied, the assemblies are formed with a 1:1:4 calmodulin/peptide/Ca 2+ stoichiometry. In contrast, the complex involving the 20‐residue binding domain of the plasma Ca 2+ pump C20W (LRRGQILWFRGLNRIQTQIK‐OH) with CaM has been shown by previous two‐dimensional nuclear magnetic resonance (2D NMR) studies to involve complexation of the C‐terminal end of CaM. Under identical conditions to those used for the amphibian peptide study, the ESI complex between C20W and CaM shows specific 1:1:2 stoichiometry. Since complex formation with the studied amphibian peptides requires Ca 2+ CaM to contain its full complement of four Ca 2+ ions, this indicates that the amphibian peptides require both ends of the CaM to effect complex formation. Charge‐state analysis and an H/D exchange experiment (with caerin 1.8) suggest that complexation involves Ca 2+ CaM undergoing a conformational change to a more compact structure. Copyright © 2008 John Wiley & Sons, Ltd.