Pukala Tara L, Urathamakul Thitima, Watt Stephen J, Beck Jennifer L, Jackway Rebecca J, Bowie John H
Department of Chemistry, The University of Adelaide, South Australia, 5005, Australia.
Rapid Commun Mass Spectrom. 2008 Nov;22(22):3501-9. doi: 10.1002/rcm.3757.
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.
抑制神经元型一氧化氮合酶（nNOS）产生一氧化氮的两栖类肽，是通过与蛋白质辅因子Ca2+钙调蛋白（Ca2+CaM）结合来实现的。使用乙酸铵水溶液缓冲系统，通过负离子电喷雾电离质谱法已证实活性肽与Ca2+CaM之间形成了复合物。在所研究的所有情况下，组装体都是以1:1:4的钙调蛋白/肽/Ca2+化学计量比形成的。相比之下，先前的二维核磁共振（2D NMR）研究表明，血浆Ca2+泵C20W的20个残基结合域（LRRGQILWFRGLNRIQTQIK-OH）与CaM形成的复合物涉及CaM C末端的络合。在与两栖类肽研究相同的条件下，C20W与CaM之间的电喷雾电离复合物显示出特定的1:1:2化学计量比。由于与所研究的两栖类肽形成复合物需要Ca2+CaM包含其全部四个Ca2+离子，这表明两栖类肽需要CaM的两端来实现复合物的形成。电荷态分析和H/D交换实验（使用caerin 1.8）表明，络合过程涉及Ca2+CaM发生构象变化，转变为更紧凑的结构。
