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

Authors

Tara L. Pukala, University of Adelaide
Thitima Urathamakul, University of WollongongFollow
Stephen James Watt, University of Wollongong
Jennifer L. Beck, University of WollongongFollow
Rebecca J. Jackway, University of Adelaide
John H. Bowie, University of Adelaide

RIS ID

25463

Publication Details

Pukala, T. L., Urathamakul, T., Watt, S. J., Beck, J. L., Jackway, R. J. & Bowie, J. (2008). Binding studies of nNOS-active amphibian peptides and Ca2+ calmodulin, using negative ion electrospray ionisation mass spectrometry. Rapid Communications in Mass Spectrometry, 22 3501-3509.

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.