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成像方法 实时的蛋白质晶体。我们将研究这一反应 由牛胰腺核糖核酸酶(RNaseA)催化 结晶态作为测试系统，因为它是一个标准 在许多开创性的实验中已经被研究过的酶 因此，分子生物学及其动力学机制 相对较好的理解。这个项目将改善我们的 理解生物学中快速过程的许多方面的能力 与人类健康中的现象有关。 晶体核糖核酸酶A具有催化活性，催化 单链RNA中3‘-5’磷酸二酯键的水解。 晶体核糖核酸酶A在乙醇水溶液中保持活性 在低温下，其反应速度很容易被 温度。通过为系统选择适当的温度， 因此，我们可以在这些晶体中选择一个反应速率 与我们数据收集方法的速度一致。我们 将在-65摄氏度将底物引入RNaseA晶体 在流动单元中，然后通过快速提高 用红外线激光测温。我们将执行这些措施 国家同步加速器X射线光源的研究 有很宽的能量带宽。由此产生的衍射象， 被称为劳厄图案，捕捉了布拉格图案的很大一部分 这块水晶的斑点。我们将重新记录同样的Laue图案 在单个事件的时间过程中连续多次 晶体内的反应事件。布拉格的变化 在反应过程中发生的强度将会产生 关于酶的动力学变化的信息 结构在反应过程中。

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.