Binding studies of nNOS-active amphibian peptides and Ca2+ calmodulin, using negative ion electrospray ionisation mass spectrometry.
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.
Department of Chemistry, The University of Adelaide, South Australia, 5005, Australia.
Tara L
TL
Pukala
Thitima
T
Urathamakul
Stephen J
SJ
Watt
Jennifer L
JL
Beck
Rebecca J
RJ
Jackway
John H
JH
Bowie
D029845:Amphibian Proteins / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001001:Anura / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D029845:Amphibian Proteins; D002147:Calmodulin; D010455:Peptides; D052248:Nitric Oxide Synthase Type I; D002118:Calcium