Department of Chemistry, University of Oxford, Physical and Theoretical Chemistry Laboratory, South Parks Road, Oxford OX1 3QZ, United Kingdom.
J Am Chem Soc. 2012 Feb 22;134(7):3429-38. doi: 10.1021/ja2096859. Epub 2012 Feb 13.
Collapse to compact states in the gas phase, with smaller collision cross sections than calculated for their native-like structure, has been reported previously for some protein complexes although not rationalized. Here we combine experimental and theoretical studies to investigate the gas-phase structures of four multimeric protein complexes during collisional activation. Importantly, using ion mobility-mass spectrometry (IM-MS), we find that all four macromolecular complexes retain their native-like topologies at low energy. Upon increasing the collision energy, two of the four complexes adopt a more compact state. This collapse was most noticeable for pentameric serum amyloid P (SAP) which contains a large central cavity. The extent of collapse was found to be highly correlated with charge state, with the surprising observation that the lowest charge states were those which experience the greatest degree of compaction. We compared these experimental results with in vacuo molecular dynamics (MD) simulations of SAP, during which the temperature was increased. Simulations showed that low charge states of SAP exhibited compact states, corresponding to collapse of the ring, while intermediate and high charge states unfolded to more extended structures, maintaining their ring-like topology, as observed experimentally. To simulate the collision-induced dissociation (CID) of different charge states of SAP, we used MS to measure the charge state of the ejected monomer and assigned this charge to one subunit, distributing the residual charges evenly among the remaining four subunits. Under these conditions, MD simulations captured the unfolding and ejection of a single subunit for intermediate charge states of SAP. The highest charge states recapitulated the ejection of compact monomers and dimers, which we observed in CID experiments of high charge states of SAP, accessed by supercharging. This strong correlation between theory and experiment has implications for further studies as well as for understanding the process of CID and for applications to gas-phase structural biology more generally.
先前已有报道称，一些蛋白质复合物在气相中会发生折叠，形成比其天然结构更小的碰撞截面的紧凑态，但这一现象尚未得到合理的解释。在此，我们结合实验和理论研究，调查了在碰撞激发过程中四个多聚体蛋白质复合物在气相中的结构。重要的是，我们使用离子淌度-质谱（IM-MS）发现，在低能量下，所有四个大分子复合物都保留了其天然的拓扑结构。当增加碰撞能时，其中两个复合物会呈现出更紧凑的状态。这种折叠在含有大中央腔的五聚体血清淀粉样蛋白（SAP）中最为明显。折叠的程度与电荷状态高度相关，令人惊讶的是，观察到电荷状态越低，其紧凑化程度越高。我们将这些实验结果与 SAP 的真空分子动力学（MD）模拟进行了比较，在模拟中，温度逐渐升高。模拟结果表明，SAP 的低电荷状态表现出紧凑态，对应于环的折叠，而中间和高电荷状态则展开为更扩展的结构，保持其环状拓扑结构，与实验观察结果一致。为了模拟 SAP 的不同电荷状态的碰撞诱导解离（CID），我们使用 MS 测量了被逐出单体的电荷状态，并将此电荷分配给一个亚基，将剩余的电荷均匀分布在其余四个亚基上。在这些条件下，MD 模拟捕捉到了 SAP 的中间电荷状态的单个亚基的展开和逐出。对于 SAP 的高电荷状态，MD 模拟再现了紧凑单体和二聚体的逐出，这与我们在 SAP 的高电荷状态 CID 实验中观察到的情况一致，这些高电荷状态是通过超荷实现的。理论与实验之间的这种强相关性对于进一步的研究以及对 CID 过程的理解以及对气相结构生物学的应用都具有重要意义。
