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

项目摘要/摘要 结构酶学的新前沿之一是从三维扩展到真正的四维- 在结构研究中加入时间维度的维度方法。而同步辐射(SR) 结晶学和低温电子显微镜可以确定它们所处的结构的细微细节 大多数情况下是在冷冻的静态样本上进行的。随着X射线自由电子激光器(XFELs)的出现， 斯坦福直线加速器相干光源(LCLS)及其“先探测后破坏”概念的发展 现在可以在接近生理条件的情况下实时跟踪酶的结构变化 在室温下(RT)。由XFELs的成功和探测器技术的最新进展和 存储环和束线设计，几个SR源也开始提供时间分辨结晶学在 RT.这些史无前例的能力将开辟新的研究领域，不仅是在生物医学领域，而且 在许多其他领域。由于这里采用的是先探测后销毁的方法，样品一般需要 在单次X光曝光后更换。因为感兴趣的生物样本通常只在稀有的 数量，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。 一种将所需样本量降至最低的连续方式。为了获得真正的分子电影 具有生物医学重要性的酶的作用，这将有助于从更深的机制上理解 这些分子机器，是必要的同步酶在被探测的样本量和启动 兴趣的反应在时间上有明确的定义。引发反应的方法可以包括混合 使用底物/化合物，或使用其他刺激，如光、温度跳跃或pH或 电势。在这项建议的框架内，我们将继续开发健壮和通用的样本 传递和反应触发方法。我们还将整合多模式检测方法，结合X射线 用互补的原位光谱技术同时探测全局结构和化学成分的衍射 酶的性质同时存在。我们将重点开发基于以下内容的按需按需方法 声学换能器技术，也探索其他液滴点胶技术和微流控技术 大大减少/消除任何样品浪费。我们将改进以前开发的原型，以 将液滴沉积在可循环和自动清洁的移动支撑物上，如磁带或轮子，用于 XFEL或SR设施的不间断连续运行。几种酶-底物混合的方法将是 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。在明确定义的酶模型系统上的实验将由 设计化学混合实验并使用测量反馈进行优化的建模方法 这个设计。这些将在SR和XFEL梁线上实施，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.