The Molecular Basis of Cellular Control Mechanisms

描述（由申请人提供）：该项目的长期目标是阐明生物系统中调控信号传递的过程，作为理解代谢控制的分子机制的一种手段。了解细胞如何调节和控制其功能的各个方面，对于我们在这些控制机制崩溃时进行干预的能力至关重要。几乎所有的信号转导模式都可以以某种方式与蛋白质构象变化有关。例如，胰岛素或胰高血糖素与其受体结合引起的构象变化调节血糖水平，或变构酶的大的四级构象变化通过改变其催化活性来调节代谢途径。在这个项目期间，我们将重点研究信号传输和变构控制的三个系统。变构酶天冬氨酸转氨基甲酰基酶（ATCase）和果糖1,6-二磷酸酶（FBPase）分别参与控制嘧啶和糖异生途径的速率。这两种酶都经历了剧烈的构象变化，包括环运动和整个蛋白质亚基的运动，以实现其功能。此外，我们将研究合作酶，二氢化酶。这种酶在嘧啶核苷酸生物合成中经历与催化活性协调的环运动。将使用各种方法来获得对这些酶如何起作用的分子水平的理解。该项目直接解决了生物信号如何传递的基本问题，特别是变构调节如何控制酶活性。该应用程序的具体目标分为基础和实际组件。我们将使用最先进的技术，包括时间分辨小角度x射线散射和时间分辨x射线晶体学，在原子水平上捕捉信号分子对这些酶系统的影响的细节。我们还将在这些酶中策略性地放置荧光探针，以监测构象变化并将这些构象变化与它们的功能联系起来。我们将使用x射线晶体学来定义ATCase的催化和变构机制的每个步骤，包括键形成的时刻。我们还将利用这些结构数据来设计高效的ATCase和FBPase抑制剂，这些抑制剂可能用于开发新的抗增殖、抗疟疾和抗糖尿病药物。了解这些蛋白质的构象变化和变构控制之间的关系也将有助于我们阐明细胞控制机制的分子基础。

DESCRIPTION (provided by applicant): The long term objectives of this project are to elucidate the processes involved in the transmission of regulatory signals in biological systems, as a means toward understanding the molecular mechanisms of metabolic control. The understanding of how cells regulate and control all aspects of their function is vital for our ability to intervene when these control mechanisms break down. Almost all modes of signal transduction can be related in some manner to protein conformational changes. For example, the conformational changes induced by the binding of insulin or glucagon to their receptors modulate blood sugar levels, or the large quaternary conformational changes of allosteric enzymes regulate metabolic pathways by altering their catalytic activity. During this project period we will concentrate on three systems involved in signal transmission and allosteric control. The allosteric enzymes aspartate transcarbamoylase (ATCase) and fructose 1,6- bisphosphatase (FBPase) are involved in controlling the rates of the pyrimidine and gluconeogenesis pathways, respectively. Both of these enzymes undergo dramatic conformational changes involving loop motions and movements of entire protein subunits for their function. In addition, we will study the cooperative enzyme, dihydroorotase. This enzyme in pyrimidine nucleotide biosynthesis undergoes loop motions that are coordinated with catalytic activity. A variety of approaches will be used to acquire a molecular-level understanding of how these enzymes function. This project directly addresses fundamental questions of how biological signals are transmitted, in general, and how allosteric regulation controls enzymatic activity, in particular. The specific aims of this application are divided into fundamental and practical components. We will use state of the art techniques involving time-resolved small-angle X-ray scattering and time-resolved X-ray crystallography to capture the details of the effects that signaling molecules have on these enzymatic systems at the atomic level. We will also use strategically placed fluorescent probes in these enzymes, both to monitor the conformational changes and to relate these conformational changes to their function. We will use X-ray crystallography to define each step in the catalytic and allosteric mechanisms of ATCase, including at the moment of bond formation. We will also use these structural data to design highly potent inhibitors of ATCase and FBPase that may be used for the development of new anti-proliferation, anti-malarial and anti-diabetic agents. Understanding of the relationship between conformational changes and allosteric control in these proteins will also help us elucidate the molecular basis of cellular control mechanisms. The understanding of the atomic level details of the transmission of regulatory signals, which control most biological processes, is critical for our ability to intervene when these controls break down. This application will concentrate on transmission of regulatory signals in enzymes involved in the pathway that produces the precursors of the nucleic acids, and of one of the pathways involved in maintaining blood sugar levels. A molecular level understanding of how these enzymes exert control over their respective pathway will provide a basis for the rational development of new anti-cancer, anti-diabetic and anti-malarial drugs.