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Type: Journal article
Title: Binding studies of nNOS-active amphibian peptides and Ca²⁺ calmodulin, using negative ion electrospray ionisation mass spectrometry
Other Titles: Binding studies of nNOS-active amphibian peptides and Ca(2+) calmodulin, using negative ion electrospray ionisation mass spectrometry
Author: Pukala, T.
Urathamakul, T.
Watt, S.
Beck, J.
Jackway, R.
Bowie, J.
Citation: Rapid Communications in Mass Spectrometry, 2008; 22(22):3501-3509
Publisher: John Wiley & Sons Ltd
Issue Date: 2008
ISSN: 0951-4198
Statement of
Tara L. Pukala, Thitima Urathamakul, Stephen J. Watt, Jennifer L. Beck, Rebecca J. Jackway, John H. Bowie
Abstract: Amphibian 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.
Keywords: Animals
Amphibian Proteins
Spectrometry, Mass, Electrospray Ionization
Binding Sites
Protein Binding
Nitric Oxide Synthase Type I
Rights: © 2008 John Wiley & Sons, Ltd.
DOI: 10.1002/rcm.3757
Grant ID: ARC
Appears in Collections:Aurora harvest
Chemistry publications

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