Detailed unfolding and folding of gaseous ubiquitin ions characterized by electron capture dissociation
Detailed unfolding and folding of gaseous ubiquitin ions characterized by electron capture dissociation
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DOI：
10.1021/ja012267j
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发表时间：
2002-06-05
影响因子：
15
通讯作者：
McLafferty, FW
中科院分区：
文献类型：
--
作者：
Breuker, K;Oh, HB;McLafferty, FW
如通过电子捕获解离（ECD）质谱法测定的，在溶液中的天然状态的泛素的解折叠焓是其气态离子的解折叠焓的5至8倍。虽然在溶液中发生两态折叠，Clemmer及其同事基于离子迁移率数据提出的三态气态过程通常得到ECD质谱的支持，包括相对产物产率，状态之间不同的DeltaH（解折叠）值，位点特异性熔化温度和折叠动力学，表明合作过程。ECD还证实，13+离子代表单独的构象异构体，可能具有侧链溶剂化的α-螺旋结构。然而，在5+至13+离子的非共价键合的ECD数据，确定总体上在69的75 interresidue网站，表明热展开的收益通过多种中间体，其构象特征也取决于电荷位点的位置。随着溶液酸度的增加，向5+离子中加入6个质子完全破坏了它们的三级非共价键。然而，新质子化位点到主链的溶剂化反而增加了这些气态离子的二级结构（可能是α-螺旋）的稳定性，而在溶液中，这些新位点通过在水性介质中的溶剂化而有助于变性。广泛的离子平衡可以导致更紧凑和多样化的构象。气态泛素的三态展开似乎涉及平行反应路径的“折叠漏斗”中的单个链构象的集合。这也提供了一个进一步的警告，从他们的气相行为表征溶液构象。
The unfolding enthalpy of the native state of ubiquitin in solution is 5 to 8 times that of its gaseous ions, as determined by electron capture dissociation (ECD) mass spectrometry. Although two-state folding occurs in solution, the three-state gaseous process proposed for this by Clemmer and co-workers based on ion mobility data is supported in general by ECD mass spectra, including relative product yields, distinct DeltaH(unfolding) values between states, site-specific melting temperatures, and folding kinetics indicating a cooperative process. ECD also confirms that the 13+ ions represent separate conformers, possibly with side-chain solvated alpha-helical structures. However, the ECD data on the noncovalent bonding in the 5+ to 13+ ions, determined overall in 69 of the 75 interresidue sites, shows that thermal unfolding proceeds via a diversity of intermediates whose conformational characteristics also depend on charge site locations. As occurs with increased acidity in solution, adding 6 protons to the 5+ ions completely destroys their tertiary noncovalent bonding. However, solvation of the newly protonated sites to the backbone instead increases the stability of the secondary structure (possibly an alpha-helix) of these gaseous ions, while in solution these new sites aid denaturation by solvation in the aqueous medium. Extensive ion equilibration can lead to even more compact and diverse conformers. The three-state unfolding of gaseous ubiquitin appears to involve ensembles of individual chain conformations in a "folding funnel" of parallel reaction paths. This also provides a further caution for characterizing solution conformers from their gas-phase behavior.