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

项目摘要/摘要 像直线加速器相干光源(LCLS)这样的X射线自由电子激光器(XFELs)的出现 斯坦福大学有可能给生物系统结构研究领域带来革命性变化。现在这是可能的 在室内使用XFELs来确定酶的结构并实时跟踪它们的反应 温度。这些前所未有的能力将开辟新的研究领域，不仅是在生物科学领域 但在其他领域也是如此。XFEL使用的一个瓶颈是缺乏一种可靠的方法来 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 在只照射一次脉冲X射线后，同时最大限度地减少生物样品的数量 使用过的，通常只有少量供应。此外，必须能够触发 反应，通过某种方法，如使用底物/化合物，或其他一些刺激，如 光，或通过温度跳跃，或电势，以便我们可以跟踪它在现实中发生的反应 时间，以便能够理解酶是如何工作的。这项提案涉及探索和 构建健壮和通用的样品传递和反应触发方法。此外，当与 补充技术，如光谱学和其他原位方法，包括全球结构和化学 可以同时获得酶的特性，从而深入了解蛋白质之间的相互作用 活性中心的结构/动力学和化学。重点发展按需点播。 方法，主要是基于声学换能器，这将大大减少或消除任何样本 浪费是更常用的基于毛细管的样品传递方法的问题，这种方法还 有其他问题，如堵塞，可以用声学液滴喷射器消除。我们建议 开发将液滴沉积在移动支撑物上的方法，例如胶带或轮子，该支撑物可以循环和 具有自动清洁功能，可在XFEL不间断连续运行。在那里，我们可以使用X射线脉冲来研究 由底物(液体/气体)激活产生的酶的中间状态，这涵盖了大部分 酶反应。其他触发方法，如光、温度跳跃或电势 这将被用于研究氧化还原活性酶系统，将被内置到我们的样品输送系统中。 将测试几种酶-底物混合的方法，重点是微米级的液液混合。 大小液滴碰撞方法，以实现更快的时间分辨率，可以随后的时间- 在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.