Excimer laser for studying ultra-rapid protein folding events

用于研究超快速蛋白质折叠事件的准分子激光器

• 批准号: 389893-2010
• 负责人: Konermann, Lars
• 金额: $ 5.41万
• 依托单位: University of Western Ontario
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments - Category 1 (<$150,000)
• 财政年份: 2009
• 资助国家: 加拿大
• 起止时间: 2009-01-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=443054
• 关键词: Excimer; laser; studying; ultra; rapid

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.

未折叠的蛋白质如何以及为什么能够自发折叠成具有生物活性的构象，一直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，这使得表征早期折叠中间体的结构极具挑战性。过去在这一领域的努力主要依赖于光谱探针，无法提供详细的蛋白质构象的见解。该提案描述了一种新的策略，将首次提供折叠最早阶段蛋白质结构的“快照”。超快速扩散混合将使变性多肽链暴露于天然条件，从而触发折叠过程。然后，受激准分子激光器将通过H2O2的光解产生OH自由基，从而在明确定义的时间点对蛋白质进行微秒级共价标记。重要的是，标记仅发生在溶剂可接近的区域，而折叠的片段受到保护。然后通过质谱法分析标记模式将得到高度详细的溶剂暴露数据，其反映折叠过程中的结构变化。2009年，我们的小组第一个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，我们现有的激光器的有限规格，目前排除了这种策略的应用超快速折叠研究。因此，我们建议购买一种新的750 Hz激光器，这将使我们的工作扩展到亚毫秒范围。这一建议代表了一个独特的机会，它将使我们的实验室率先探索超快速折叠事件与迄今无法获得的结构分辨率。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。如果不能为这种设备获得资金，我们的研究计划将受到严重挫折。