Excimer laser for studying ultra-rapid protein folding events

如何以及为什么展开的蛋白质可以自发折叠成具有生物活性的构象，这一问题仍然是生物化学中尚未解决的主要问题之一。一个特别重要的瓶颈是缺乏关于折叠最早阶段的信息。这些超快速的过程发生在亚毫秒级的时间尺度上，这使得表征早期折叠中间体的结构变得非常具有挑战性。过去在这一领域的努力主要依赖于光谱探测器，而光谱探测器无法提供对蛋白质构象的详细了解。这项提议描述了一种新的策略，它将首次在折叠的最早阶段提供蛋白质结构的“快照”。超快速扩散混合会将变性多肽链暴露在自然条件下，从而触发折叠过程。然后，准分子激光将通过光解过氧化氢产生羟基自由基，从而在明确定义的时间点对蛋白质进行微秒共价标记。重要的是，标记只发生在溶剂可及区域，而折叠片段受到保护。通过质谱学对标记模式的分析将得到非常详细的溶剂暴露数据，这些数据反映了折叠过程中的结构变化。我们小组在2009年率先证明了这种方法的可行性，尽管时间尺度相对较慢。不幸的是，我们现有激光器的有限规格目前阻碍了这一策略在超快折叠研究中的应用。因此，我们提议购买一台新的750赫兹激光器，这将使我们的工作扩展到亚毫秒范围。这一提议代表着一个独特的机会；它将使我们的实验室率先探索具有迄今无法获得的结构分辨率的超快折叠事件。由此产生的见解将加强对生物自组装以及可能导致疾病状态的异常副反应的总体理解。这些研究活动将为HQP培训提供极好的机会。如果不能为这种设备获得资金，将是我们研究计划的严重挫折。

The question how and why unfolded proteins can spontaneously fold to their biologically active conformations remains one of the major unsolved problems in biological chemistry. One particularly important bottleneck is the lack of information regarding the very earliest stages of folding. These ultra-rapid processes occur on a sub-millisecond time scale, which makes it exceedingly challenging to characterize the structures of early folding intermediates. Past efforts in this area have largely relied on spectroscopic probes that are unable to supply detailed insights into protein conformations. This proposal describes a novel strategy that will for the first time provide "snapshots" of protein structures during the very earliest stages of folding. Ultra-rapid diffusive mixing will expose denatured polypeptide chains to native conditions, thereby triggering the folding process. An excimer laser will then produce OH radicals by photolysis of H2O2, resulting in microsecond covalent labeling of the protein at well defined time points. Importantly, labeling only occurs in solvent-accessible regions, whereas folded segments are protected. Analyses of the labeling pattern by mass spectrometry will then result in highly detailed solvent exposure data which reflect the structural changes during folding. Our group was the first to demonstrate the viability of such an approach in 2009, albeit on relatively slow time scales. Unfortunately, the limited specifications of our existing laser currently preclude the application of this strategy to ultra-rapid folding studies. We therefore propose the acquisition of a new 750 Hz laser that will allow an extension of our work to the sub-millisecond range. This proposal represents a unique opportunity; it will allow our laboratory to pioneer the exploration of ultra-rapid folding events with hitherto unobtainable structural resolution. The resulting insights will enhance the general understanding of biological self-assembly, as well as aberrant side reactions that can lead to disease states. These research activities will provide superb opportunities for HQP training. Failure to secure funding for this equipment would be a severe setback for our research program.