A Model System for Sensor Proteins

描述（由申请人提供）：光敏黄蛋白（PYP）是一个大家族蛋白质的原型，该家族含有一个称为PAS结构域的结构基序。PAS结构域在真核生物和原核生物中具有多种信号传导和调节作用。光活性黄蛋白（PYP）是紫色细菌中一种可溶性小分子蛋白，具有多种信号转导功能。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向其反应伴侣发出信号并利用光循环在体内发挥作用的机制。4.确定迄今为止确定的三种类型PYP在功能和物理化学性质方面的差异。将研究野生型、位点特异性突变体和变体发色团的结构、热力学、动力学和光学性质。将利用各种技术，包括X射线晶体学，NMR，UV/维斯光谱，从飞秒到秒的时域动力学测量，皮秒相干反斯托克斯拉曼光谱（汽车），皮秒时间分辨汽车和计算机建模。首先，PYP将提供关键的见解，这将有助于阐明PAS结构域在信号传导和调节中的作用。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提供了了解光活性蛋白质中关键元素的机会-基态蛋白质和关键中间体的光谱红移，光异构化如何用于驱动功能，以及光循环中间体的结构。这种理解水平将补充膜结合光敏蛋白（细菌视紫红质，感觉视紫红质和视紫红质）的工作，并将提供这些系统无法提供的信息。第三，对PYP和光敏蛋白的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、低功率电子器件中应用

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