NANOSECOND TEMPERATURE-JUMP SAXS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 世界范围内都在努力尝试从蛋白质的序列中预测蛋白质的折叠。为了补充这些计算的努力，一些技术被用来确定在这个过程中的中间体，并测量这些中间状态之间的变化的动力学。这些方法的时间分辨率是关键，因为它限制了它们在鉴定蛋白质折叠中间体中的实用性。温度跳跃技术受到越来越多的关注，因为高功率激光器的可用性，它可以均匀地突然增加小样本的水温几十摄氏度，而无需使用添加的染料或其他吸收剂。这种方法已与几种时间分辨光学技术相结合，研究蛋白质折叠，催化和反应动力学。不幸的是，可见光探针，如荧光或吸收光谱产生的局部结构信息量有限。相比之下，SAXS提供了关于蛋白质在溶液中的全局形状变化的信息。然而，目前的方法可能仍然限于~100微秒的时间分辨率，因此无法测量最早和最快的折叠事件，例如那些参与蛋白质下坡折叠或疏水性塌陷的过程，其中整个过程可能仅需要几十微秒或更少。我们打算规避现有技术固有的死区时间和信噪比限制，以达到亚稳态。微秒的时间尺度的解决方案SAXS。在过去的一年里，我们已经估计了暴露时间，建立了仪器，并充分表征了我们将用于未来时间分辨测量的蛋白质系统的静态行为。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. There is a worldwide effort to try to predict protein folds from their sequences. To supplement these computational efforts, a number of techniques are used to identify the intermediates in this process and measure the kinetics of the changes between these intermediate states. The time resolution of these methods is key, as it limits their utility in identifying protein folding intermediates. Temperature-jump techniques have received increasing attention because of the availability of high-power lasers that can uniformly and suddenly increase the water temperature of a small sample tens of degrees Celsius without the use of added dyes or other absorbers. This method has been combined with several time-resolved optical techniques to study protein folding, catalysis, and reaction kinetics. Unfortunately, visible light probes such as fluorescence or absorption spectroscopies yield a limited amount of local structural information. In contrast, SAXS provides information on global shape changes of a protein in solution. However, present methods are likely to remain limited to ~100 microsecond temporal resolution and have therefore been unable to measure the earliest and fastest folding events, such as those in involved in downhill protein folding or hydrophobic collapse where the entire process may take only a few tens of microseconds or less. We intend to circumvent the inherent deadtime and signal-to-noise limitations of existing techniques to reach sub-microsecond timescales in solution SAXS. In the past year we have estimated exposure times, built instrumentation, and fully characterized the static behavior of the protein systems we will be using for future time-resolved measurements.