High throughput sample delivery method for time resolved studies of enzyme reactions with X-ray and complementary techniques

项目概要/摘要 结构酶学的新领域之一是从三维扩展到真正的四维， 维度方法，在结构研究中添加时间维度。同步辐射（SR） 晶体学和低温电子显微镜可以测定它们所处的微小细节的结构。 大多数情况下进行冷冻静态样品。随着X射线自由电子激光器（XFEL）的出现， 斯坦福大学的直线加速器相干光源（LCLS），以及“探测前破坏”概念的发展， 现在有可能在接近生理条件的真实的时间内跟踪酶的结构变化 在室温（RT）下反应。在XFEL的成功和探测器技术的最新进展的推动下， 存储环和束线设计，几个SR源也开始提供时间分辨晶体学， RT.这些前所未有的能力将开辟新的研究领域，不仅在生物医学科学， 在许多其他领域。由于这里使用的“破坏前探测方法”，样品通常需要 在单次X射线曝光后更换。由于感兴趣的生物样品通常只能在稀缺的 量，必须开发一种稳健的方法，将样品引入X射线相互作用区域， 最小化所需样品量的连续方式。为了获得真正的“分子电影”， 酶的生物医学重要性的行动，这将有助于更深层次的机械理解， 在这些分子机器中，必须使探针样品体积中的酶同步，并启动 以时间上明确定义的方式进行感兴趣的反应。用于反应引发的方法可包括混合 与底物/化合物接触，或利用其他刺激，如光、温度突变或pH变化，或 电势在此建议的框架内，我们将继续开发强大的和通用的样本 传递和反应触发方法。我们还将整合多模态检测方法，结合X射线 用互补的原位光谱技术来探测全局结构和化学性质 酶的特性。我们将重点开发基于以下方面的按需投放方法： 声换能器技术，而且还探索其他液滴分配技术和微流体技术， 基本上减少/消除任何样品浪费。我们将改进先前开发的原型， 将所述液滴沉积在可循环且自清洁的移动支撑件（例如带或轮）上， XFEL或SR设施的不间断连续运行。将介绍几种酶-底物混合的方法。 测试，重点是液-气和液-液混合，包括微米级液滴碰撞方法 以实现更快的时间分辨率。在定义明确的酶模型系统上进行的实验将由 设计化学混合实验的建模方法，并使用测量反馈来优化 设计。这些将在SR和XFEL光束线上实施，并可用于更广泛的结构 酶学用户社区。

Project Summary/Abstract One of the new frontiers in structural enzymology is the expansion from a three-dimensional to a truly four- dimensional approach by adding the time dimension to structural studies. While Synchrotron Radiation (SR) crystallography and cryo Electron Microscopy allow the determination of structures in minute detail they are in most cases performed on frozen static samples. With the advent of X-ray free electron lasers (XFELs) like the Linac Coherent Light Source (LCLS) at Stanford, and the development of the “probe before destroy” concept it now is possible to follow structural changes in enzymes in real time and under close to physiological conditions at room temperature (RT). Driven by the success of XFELs and recent advances in detector technology and storage ring and beam line design, several SR sources are also starting to offer time resolved crystallography at RT. These unprecedented capabilities will open new fields of research, not only in biomedical sciences but also in many other areas. Due to the “probe before destroy approach” utilized here, the samples generally need to be replaced after a single X-ray exposure. As biological samples of interest are often only available in scarce amounts, it is mandatory to develop a robust method to introduce the sample into the X-ray interaction region in a continuous manner that minimizes the required sample amount. In order to obtain true “molecular movies” of enzymes of biomedical importance in action, which will contribute to a deeper mechanistic understanding of these molecular machines, it is essential to synchronize the enzyme in the probed sample volume and initiate the reaction of interest in a temporally well-defined manner. Methods for reaction initiation can include mixing with a substrate/chemical compound, or utilize other stimuli such as light, temperature jump, or change in pH or electrical potential. In the frame of this proposal, we will continue the development of robust and versatile sample delivery and reaction triggering methods. We will also integrate multi-modal detection methods, combining X-ray diffraction with complementary in situ spectroscopic techniques to probe both global structures and chemical properties of enzymes concurrently. We will focus on the development of drop-on-demand methods based on acoustic transducer technology, but also explore other droplet dispensing technologies and microfluidics to substantially diminish/eliminate any sample wastage. We will improve the previously developed prototypes for depositing the drops on a moving support, such as a tape or wheel, that can circulate and is self-cleaning, for non-stop continuous operation at the XFEL or SR facility. Several methods for enzyme-substrate mixing will be tested, with emphasis on liquid-gas and liquid-liquid mixing, including with micron size droplet collision methods to achieve faster time resolution. Experiments on well-defined enzyme model systems will be accommodated by modeling approaches to design chemical mixing experiments and use feedback from measurements to optimize the design. These will be implemented at SR and XFEL beam lines and made available for the broader structural enzymology user community.