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

项目概要/摘要 X射线自由电子激光器（XFEL）的出现，如直线加速器相干光源（LCLS）， 斯坦福大学有可能彻底改变生物系统结构研究领域。现在可以 使用XFEL来确定酶的结构，并在室温下真实的时间内跟踪它们的反应， 温度这些前所未有的能力将开辟新的研究领域，不仅在生物科学领域， 而且在其他领域也是如此。使用XFEL的一个瓶颈是缺乏一种可靠的方法， 当样品被破坏时，以连续的方式将样品引入X射线相互作用区域 在暴露于X射线的一个脉冲之后，同时最小化生物样品的量， 使用，这往往是只有在小数量。此外，必须能够触发 反应，通过某种方法，如使用底物/化合物，或一些其他刺激，如 光，或者温度的跳跃，或者电势，这样我们就可以跟踪真实的反应 时间，以便能够了解酶的功能。该提案涉及探索和 构建稳健且通用的样品递送和反应触发方法。此外，当与 补充技术，如光谱学和其他方法在现场，无论是全球结构和化学 酶的性质可以同时获得，提供了对蛋白质之间的相互作用的见解， 结构/动力学和化学活性位点。我们将重点发展按需点播 方法，主要是基于声换能器，这将大大减少或消除任何样品， 浪费是更常用的基于毛细管的样品输送方法的问题， 还存在其它问题，例如通过声学液滴喷射器消除的堵塞。我们建议 开发用于将液滴沉积在可循环的移动支撑物（例如带或轮）上的方法， 是自清洁的，用于XFEL的不间断连续操作。在那里我们可以用X射线脉冲来研究 酶中的中间状态，将由底物（液体/气体）活化产生，这涵盖了大多数酶的活性。 酶反应。其他触发方法，如光、温度突变或电势 将用于研究氧化还原活性酶系统，将内置到我们的样品输送系统中。 将测试几种用于酶-底物混合的方法，重点是用微米进行液-液混合 尺寸液滴碰撞方法，以实现更快的时间分辨率，可以随后的时间- X射线探测器之前的进化

Project Summary/Abstract The advent of the X-ray free electron lasers (XFELs) like the Linac Coherent Light Source (LCLS) at Stanford has the potential to revolutionize the field of structural studies of biological systems. It is now possible using the XFELs to determine the structures of enzymes and follow their reactions in real time, at room temperature. These unprecedented capabilities will open new fields of research, not only in biological sciences but also in other areas. One bottle-neck in the use of the XFELs has been the lack of a robust method to introduce the sample into the X-ray interaction region in a continuous manner as the samples are destroyed after exposure to just one pulse of X-rays, and at the same time minimize the amount of biological sample used, which are often only available in small quantities. Moreover, it is imperative to be able to trigger the reactions, by some method, such as using a substrate/chemical compound, or some other stimulus such as light, or by a temperature jump, or electrical potential so that we can follow the reaction as it happens in real time, in order to be able to understand how the enzyme functions. This proposal deals with exploring and constructing robust and versatile sample delivery and reaction triggering methods. Also, when combined with complementary techniques like spectroscopy and other methods in situ, both global structures and chemical properties of enzymes can be obtained concurrently, providing insights into the interplay between the protein structure/dynamics and chemistry at an active site. We will focus on the development of drop-on-demand methods, mostly based on an acoustic transducer, that will substantially diminish or eliminate any sample wastage which is a problem with the more commonly used capillary based sample delivery methods that also have other problems such as clogging that are eliminated with an acoustic droplet ejector. We propose to develop methods 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. There we can use the X-ray pulses to study the intermediate states in enzymes that will be generated by substrate (liquid/gas) activation, which covers most of the enzymatic reactions. Other triggering methods such as light, or temperature jump, or an electric potential that will be used to study redox active enzyme systems, will be built-in into our sample delivery system. Several methods for enzyme-substrate mixing will be tested, with emphasis on liquid-liquid mixing with micron size droplet collision methods to achieve faster time resolution that can be followed by subsequent time- evolution before the X-ray probe.