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首页> 外文期刊>Biochemistry >Calcium-induced structural transitions of the calmodulin-melittin system studied by electrospray mass spectrometry: Conformational subpopulations and metal-unsaturated intermediates
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Calcium-induced structural transitions of the calmodulin-melittin system studied by electrospray mass spectrometry: Conformational subpopulations and metal-unsaturated intermediates

机译：钙诱导的钙调蛋白-褪黑素系统的结构转变，通过电喷雾质谱研究：构象亚群和金属不饱和中间体

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摘要

Calmodulin (CaM) is a calcium-sensing protein that can bind to and activate various target enzymes. Here, electrospray ionization mass spectrometry (ESI-MS) was used to investigate calcium-induced structural changes of CaM, as well as binding to the model target melittin (Mel). Nonspecific metalation artifacts were eliminated by conducting the experiments in negative ion mode and with calcium tartrate as metal source [Pan et al. (2009) Anal. Chem. 81, 5008]. Two coexisting CaM subpopulations can be distinguished on the basis of their ESI charge state distributions, namely, relatively disordered conformers (CaM_D, high charge states) and more tightly folded proteins (CaM_F, low charge states). Calcium titration experiments on isolated CaM reveal that the transition from apo-CaM_D to Ca_4· CaM_F proceeds with apparent K_d values of 10, 14, 30, and 12 μM. In the presence of Mel, a gradual [Ca~(2+)] increase results in an overall population shift from apo-CaM_D to Ca_4·CaM_F·Mel. This transition involves various intermediates, Ca_n·CaM_F·Mel with n = 0,..., 3, as well as apo-CaM_D·Mel. Thus, neither the binding of four Ca~(2+) nor the existence of a tightly folded CaM conformation is a prerequisite for target binding. Millisecond time-resolved ESI-MS experiments were conducted to monitor the response of a premixed CaM-Mel solution to a calcium concentration jump, thereby mimicking the conditions encountered in a cellular signaling context. The resulting data suggest that the formation of Ca_4·CaM_F·Mel proceeds along three parallel kinetic pathways: (1) metal binding to CaM_D followed by formation of a compact protein-target complex, (2) folding of the apoprotein, then target binding, followed by metal complexation, (3) target binding to apo-CaM D followed by sequential metal binding. The exact structural properties of the various metal-unsaturated CaM species, as well as their physiological roles, remain to be elucidated.
机译：钙调蛋白（CaM）是一种钙敏感蛋白，可以结合并激活各种目标酶。在这里，电喷雾电离质谱（ESI-MS）用于研究钙诱导的CaM的结构变化，以及与模型目标蜂毒素（Mel）的结合。通过在负离子模式下进行实验并使用酒石酸钙作为金属源，可以消除非特异性金属化伪影[Pan等。 （2009）肛门。化学81，5008]。可以根据其ESI电荷状态分布来区分两个共存的CaM亚群，即相对无序的构象异构体（CaM_D，高电荷状态）和折叠紧密的蛋白质（CaM_F，低电荷状态）。在分离的CaM上进行钙滴定实验表明，从apo-CaM_D到Ca_4·CaM_F的转变以10、14、30和12μM的表观K_d值进行。在Mel的存在下，[Ca〜（2+）]的逐渐增加导致总体人口从apo-CaM_D转移到Ca_4·CaM_F·Mel。该转变涉及各种中间体，n = 0，...，3的Ca_n·CaM_F·Mel以及apo-CaM_D·Mel。因此，四个Ca〜（2+）的结合或紧密折叠的CaM构象的存在都不是靶标结合的先决条件。进行了毫秒时间分辨的ESI-MS实验，以监控预混合的CaM-Mel溶液对钙浓度跃迁的响应，从而模拟细胞信号环境中遇到的情况。所得数据表明，Ca_4·CaM_F·Mel的形成沿着三个平行的动力学路径进行：（1）金属与CaM_D结合，然后形成紧密的蛋白质-靶标复合物;（2）载脂蛋白折叠，然后与靶标结合，继之以金属络合，（3）靶标与apo-CaM D的结合，然后依次进行金属结合。各种金属不饱和CaM种类的确切结构性质及其生理作用仍有待阐明。

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