LAUE DIFFRACTION STUDY OF BOVINE PANCREATIC RIBONUCLEASE

我们建议系统地发展劳厄衍射 酶催化的快速时间分辨率成像方法 蛋白质晶体在真实的时间。 我们将研究 由牛胰腺核糖核酸酶（RNase A）催化， 晶体状态作为测试系统，因为它是一个标准 在许多开创性的实验中， 分子生物学及其动力学机制， 相对比较好理解。 该项目将改善我们的 能够理解生物学中快速过程的许多方面 与人类健康有关的现象。 晶体RNA酶A具有催化活性，催化 单链RNA中3 '-5'磷酸二酯键的水解。 晶体RNA酶A在醇水溶液中保持活性 在低温下，它的反应速率很容易调节， 温度 通过为系统选择适当的温度， 因此，我们可以选择这些晶体中的反应速率， 与我们数据收集方法的速度一致。 我们 将在-65 ℃下将底物引入RNase A晶体中 在流动池中，然后通过快速升高 用红外线激光加热。 我们将执行这些 在国家同步加速器光源上对X射线束的研究 能量带宽很宽 得到的衍射图像， 称为劳厄模式，捕捉了布拉格的大部分， 找到了这块水晶 我们将重新记录同样的劳厄模式 在一个单一的时间过程中连续多次 晶体内的反应事件。 布拉格的变化 在反应过程中出现的强度将产生 关于酶中发生的动力学变化的信息 反应过程中的结构。

We propose to systematically develop Laue diffraction methods for fast time-resolution imaging of enzyme catalysis in protein crystals in real time. We will study the reaction catalyzed by bovine pancreatic ribonuclease (Rnase A) in the crystalline state as a test system because it is a standard enzyme that has been studied in many pioneering experiments in molecular biology and its kinetic mechanism is therefore relatively well understood. This project will improve our ability to understand many aspects of fast processes in biology that relate to phenomena in human health. Crystalline Rnase A is catalytically active, catalyzing the hydrolysis of 3'-5' phosphodiester bonds in single-stranded RNA. Crystalline Rnase A remains active in aqueous alcohol solutions at low temperatures, and its reaction rate is easily modulated by temperature. By selecting a proper temperature for the system, we can therefore choose a reaction rate in these crystals that is consistent with the speed of our data collection methods. We will introduce substrate into Rnase A crystals at -65 degrees C in a flow cell, then initiate the reaction by quickly raising the temperature with an infrared laser. We will carry out these studies at the National Synchrotron Light Source on an x-ray beam with a wide energy bandwidth. The resulting diffraction image, called a Laue pattern, captures a large fraction of the Bragg spots for this crystal. We will re-record this same Laue pattern many times sequentially during the time course of a single reaction event within the crystal. Changes in the Bragg intensities that occur during the reaction will then yield information about kinetic changes occurring in the enzyme structure during the reaction.