Folding and structural dynamics of proteins studied by electrospray mass spectrometry

我们的研究旨在阐明蛋白质如何折叠，它们如何与其他分子相互作用，以及为什么某些条件会导致错误折叠和疾病。这项工作大部分依赖于电喷雾质谱（ESI-MS）的使用。“时间分辨”ESI-MS是我们实验室开发的一项技术，可以在毫秒到秒的时间尺度上监测折叠过程中蛋白质致密度的变化。同时，氢键的整体水平，以及与非共价结合伙伴（如其他蛋白质）的相互作用可以被探测。我们的目标之一是利用各种策略，在以前无法达到的水平上表征短寿命折叠中间体的结构。一种特别有前途的方法是在溶液中脉冲氢交换后，在气相中使用电子捕获解离来分解蛋白质。通过测量单个片段的交换水平，将有可能以空间分辨的方式阐明氢键形成的时间顺序。水动力聚焦的使用将大大提高时间分辨率，低至约1毫秒，这样甚至可以检测到非常早期的折叠步骤。同时，我们将采用光化学方法对蛋白质进行微秒共价标记。以这种方式获得的信息（侧链的表面暴露）将与从同位素交换实验（氢键）获得的信息相补充。最终，这些活动将允许“分子电影”的发展，描绘折叠过程中构象变化的完整序列。在相关工作中，快速氢交换将用于研究酶的构象动力学和活性之间的联系。这些实验将为长期存在的问题提供答案，即酶的内部运动是否在周转过程中增强，或者催化是否在静息状态的内在动力学给出的限制内发生。计算机模拟将揭示在ESI过程中蛋白质离子释放到气相的确切机制。所有这些活动都将为HQP培训提供极好的机会。

Our research aims to elucidate how proteins fold, how they interact with other molecules, and why certain conditions lead to misfolding and disease. Much of this work relies on the use of electrospray mass spectrometry (ESI-MS). "Time-resolved" ESI-MS, a technique developed in our laboratory, allows changes in protein compactness during folding to be monitored on a time-scale of milliseconds to seconds. Simultaneously, the overall level of hydrogen bonding, as well as interactions with noncovalent binding partners (such as other proteins) can be probed. One of our goals is to characterize the structure of short-lived folding intermediates at a previously unattainable level, using a variety of strategies. A particularly promising approach is the use of electron capture dissociation for the fragmentation of proteins in the gas phase, following pulsed hydrogen-exchange in solution. By measuring the exchange levels of individual fragments it will become possible to elucidate the temporal order of hydrogen bond formation in a spatially-resolved manner. The use of hydrodynamic focusing will provide dramatic improvements in time resolution down to around one millisecond, such that even very early folding steps can be detected. In parallel, we will employ photochemical methods for the microsecond covalent labeling of proteins. The information obtained in this way (surface exposure of side chains) will be complementary to that obtained from isotope exchange experiments (hydrogen bonding). Ultimately, these activities will allow the development of "molecular movies", depicting the complete sequence of conformational changes during folding. In related work, rapid hydrogen exchange will be used to investigate the link between enzyme conformational dynamics and activity. These experiments will provide an answer to the long-standing question if the internal motions of enzymes are enhanced during turnover, or whether catalysis occurs within the limitations given by the intrinsic dynamics of the resting state. Computer simulations will shed light on the exact mechanism by which protein ions are released into the gas phase during ESI. All of these activities will provide excellent opportunities for HQP training.