Excimer laser for studying ultra-rapid protein folding events

未折叠的蛋白质如何以及为什么能自发折叠成具有生物活性的构象仍然是生物化学中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的时间尺度上，这使得描述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很大程度上依赖于光谱探针，而光谱探针无法提供蛋白质构象的详细信息。这一提议描述了一种新颖的策略，将首次提供折叠最早期阶段蛋白质结构的“快照”。超快速扩散混合会使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H2O2光解产生OH自由基，从而在确定的时间点对蛋白质进行微秒级共价标记。重要的是，标记只发生在溶剂可接近的区域，而折叠的片段是受保护的。然后，通过质谱分析标签模式将产生非常详细的溶剂暴露数据，反映折叠过程中的结构变化。我们的小组在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种策略在超快速折叠研究中的应用。因此，我们建议购买一种新的750赫兹激光器，这将使我们的工作扩展到亚毫秒范围。这个提议代表了一个独特的机会；这将使我们的实验室率先探索迄今为止无法获得的结构分辨率的超快速折叠事件。由此产生的见解将增强对生物自组装的一般理解，以及可能导致疾病状态的异常副反应。这些研究活动将为HQP培训提供极好的机会。如果不能为这台设备筹措资金，我们的研究计划将受到严重挫折。

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.