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Binding studies of nNOS-active amphibian peptides and Ca²⁺ calmodulin, using negative ion electrospray ionisation mass spectrometry

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dc.contributor.authorPukala, T.
dc.contributor.authorUrathamakul, T.
dc.contributor.authorWatt, S.
dc.contributor.authorBeck, J.
dc.contributor.authorJackway, R.
dc.contributor.authorBowie, J.
dc.date.issued2008
dc.description.abstractAmphibian peptides which inhibit the formation of nitric oxide by neuronal nitric oxide synthase (nNOS) do so by binding to the protein cofactor, Ca2+calmodulin (Ca2+CaM). Complex formation between active peptides and Ca2+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/Ca2+ stoichiometry. In contrast, the complex involving the 20-residue binding domain of the plasma Ca2+ 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 Ca2+CaM to contain its full complement of four Ca2+ 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 Ca2+CaM undergoing a conformational change to a more compact structure.
dc.description.statementofresponsibilityTara L. Pukala, Thitima Urathamakul, Stephen J. Watt, Jennifer L. Beck, Rebecca J. Jackway, John H. Bowie
dc.identifier.citationRapid Communications in Mass Spectrometry, 2008; 22(22):3501-3509
dc.identifier.doi10.1002/rcm.3757
dc.identifier.issn0951-4198
dc.identifier.issn1097-0231
dc.identifier.orcidPukala, T. [0000-0001-7391-1436]
dc.identifier.urihttp://hdl.handle.net/2440/53520
dc.language.isoen
dc.publisherJohn Wiley & Sons Ltd
dc.relation.grantARC
dc.rights© 2008 John Wiley & Sons, Ltd.
dc.source.urihttps://doi.org/10.1002/rcm.3757
dc.subjectAnimals
dc.subjectAnura
dc.subjectCalcium
dc.subjectPeptides
dc.subjectAmphibian Proteins
dc.subjectCalmodulin
dc.subjectSpectrometry, Mass, Electrospray Ionization
dc.subjectBinding Sites
dc.subjectProtein Binding
dc.subjectNitric Oxide Synthase Type I
dc.titleBinding studies of nNOS-active amphibian peptides and Ca²⁺ calmodulin, using negative ion electrospray ionisation mass spectrometry
dc.title.alternativeBinding studies of nNOS-active amphibian peptides and Ca(2+) calmodulin, using negative ion electrospray ionisation mass spectrometry
dc.typeJournal article
pubs.publication-statusPublished

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