A Model System for Sensor Proteins

描述(由申请人提供)：光活性黄色蛋白(PYP)是一个包含结构基序的蛋白质大家族的原型，称为PAS结构域。PAS结构域在真核生物和原核生物中发挥着不同的信号和调节作用。光活性黄色蛋白(PYP)是一种小分子可溶性蛋白，在紫色细菌中具有多种信号转导作用。拟议研究的目标有四个方面。1.阐明光循环中间体的化学和结构特征。2.了解特定氨基酸侧链在基态和中间体的动力学和光谱性质中的作用。3.建立PYP向其反应伙伴传递信号并利用光循环在体内发挥作用的机制(S)。4.确定迄今已鉴定的三种类型的PYP在功能和理化性质上的差异。我们将研究野生型、特定位点突变体和变异型发色团的结构、热力学、动力学和光学性质。将利用各种技术，包括X射线结晶学、核磁共振、UV/Vis光谱、从飞秒到秒的时间域的动力学测量、皮秒相干反斯托克斯拉曼光谱(CARS)、皮秒时间分辨CARS和计算机模拟。首先，PYP将提供关键的见解，这将有助于阐明PAS结构域在信号和调控中的作用。这些信息将直接有助于更好地了解PAS域家族的所有成员。其次，PYP提供了机会来理解光活性蛋白质中的关键元素-基态蛋白质和关键中间体的光谱红移，如何利用光异构化来驱动功能，以及光循环中间体的结构。这一水平的理解将补充与膜结合的光活性蛋白(细菌视紫红质、感觉性视紫红质和视紫红质)的工作，并将提供这些系统无法获得的信息。第三，对PYP和光活性蛋白质的总体理解可能有助于光生物电子开关器件的开发，该器件将在下一代小型、快速、低功率电子设备中应用

DESCRIPTION (provided by applicant): Photoactive yellow protein (PYP) is the prototype for a large family of proteins containing a structural motif referred to as the PAS domain. PAS domains function in a variety of signaling and regulatory roles in eucaryotes as well as procaryotes. Photoactive yellow protein (PYP) is a small soluble protein which appears to have a variety of signaling roles in purple bacteria. The goals of the proposed studies are four fold. 1. To elucidate the chemical and structural characteristics of the photocycle intermediates. 2.To understand the role of specific amino acid side chains in the kinetic and spectral properties of the ground state and intermediates. 3. To establish the mechanism(s) by which PYP signals to its reaction partners and utilizes the photocycle to function in vivo. 4. To establish the differences, in both functional and physico-chemical properties, among the three types of PYP identified to date. Structural, thermodynamic, kinetic and optical properties of the wild-type, site-specific mutants and variant chromophores will be studied. A variety of techniques will be utilized, including x-ray crystallography, NMR, UV/VIS spectroscopy, kinetic measurements in time domains ranging from femtoseconds to seconds, picosecond Coherent Anti-Stokes Raman Spectroscopy (CARS), picosecond time resolved CARS, and computer modeling.The significance of the proposed work is three fold. First, PYP will provide key insights which will help elucidate the role of PAS domains in signaling and regulation. This information will contribute directly to a better understanding of all of the members of the PAS domain family. Second, PYP offers the opportunity to understand the key elements in photoactive proteins - the spectral red-shift of the ground state protein and of key intermediates, how photoisomerization can be used to drive function, and the structure of photocycle intermediates. This level of understanding will complement work with membrane-bound photoactive proteins (bacteriorhodopsin, sensory rhodopsin and rhodopsin) and will provide information not available with these systems. Third, it is possible that an understanding of PYP and photoactive proteins in general will contribute to the development of photobioelectronic switching devices which will have applications in the next generation of small, fast, low power electronics