The thermal unfolding of native cytochrome c in the transition from solution to gas phase probed by native electron capture dissociation
The thermal unfolding of native cytochrome c in the transition from solution to gas phase probed by native electron capture dissociation
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DOI：
10.1002/anie.200500668
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发表时间：
2005-01-01
影响因子：
16.6
通讯作者：
McLafferty, FW
中科院分区：
文献类型：
--
作者：
Breuker, K;McLafferty, FW
水合作用对蛋白质结构、动力学、折叠和稳定性的影响已经研究了几十年，仍然是一个活跃的研究课题。[1-4]去除溶剂会导致形成稳定的气相蛋白质构象，这种构象可能与原始的自然状态有很大的不同，[5-9]但到目前为止，人们对突然暴露在真空中的天然蛋白质的结构变化序列一无所知。我们在这里讨论了水合作用对结构和稳定性的影响，并首次报道了天然蛋白质结构在从溶液到气相的转变过程中的热展开数据。我们的数据支持一种以失去疏水键为主的顺序去折叠机制。我们最近已经表明，在有利于形成非共价结合的同源二聚体(%75μm)的浓度下，铁细胞色素c(FeIII)-Cytc的水溶液的电喷雾电离[10]会产生意想不到的主干-裂解产物，我们将这种现象称为“天然电子捕获解离”(Necd)。[11]简单地说，当一个电喷雾二聚体被引入傅里叶变换质谱仪(FTMS)并通过加热的毛细管去溶时，它的一个单体部分展开。这会导致质子从致密的单体II转移到部分展开的单体I，并导致很大的电荷不对称。[12]这反过来又促使两个电子在分子间转移到单体I的血红素，一个减少了血红素铁，另一个导致蛋白质骨架裂解(NECD)，紧接着与血红素接触的残基。[11，13]最后，两个裂解
The effect of hydration on protein structure, dynamics, folding, and stability has been studied for decades and still is a topic of active research.[1–4] Removal of solvent results in the formation of stable gas-phase protein conformations that can be dramatically different from the original native state,[5–9] but so far nothing is known about the sequence of structural changes of a native protein that is suddenly exposed to vacuum. We address here the effect of hydration on structure and stability, and report for the first time site-specific data on the thermal unfolding of a native protein structure in the transition from solution to gas phase. Our data support a sequential unfolding mechanism dominated by the loss of hydrophobic bonding.We have shown recently that electrospray ionization (ESI)[10] of aqueous solutions of ferric Cytochrome c,(FeIII)-Cytc, in concentrations favorable for the formation of noncovalently bound homodimers (% 75 μm) produces unexpected backbone-cleavage products, a phenomenon we termed “native electron capture dissociation”(NECD).[11] Briefly, as an electrosprayed dimer ion is introduced into the Fourier transform mass spectrometer (FTMS) and passes the heated capillary for desolvation, one of its monomers partially unfolds. This causes proton transfer from the compact monomer II to the partially unfolded monomer I, and induces a substantial charge asymmetry.[12] In turn, this prompts intermolecular transfer of two electrons to the heme of monomer I, one reducing the heme iron and the other causing protein backbone cleavage (NECD) next to residues in contact with the heme.[11, 13] Finally, the two cleavage