Advanced Infrared Biology of Protein Structure & Dynamics

4.项目摘要/摘要 蛋白质中的质子转移是蛋白质基本功能的一个基本过程，包括 信号/调节、生物能量学和生物催化。了解什么会触发质子转移和 质子转移如何驱动后续功能上重要的结构转变具有重大意义 了解许多蛋白质的结构-功能关系的重要性。这样的研究意义重大 由于缺乏可广泛使用的探测质子动态变化的结构技术 蛋白质功能过程中的位置。该项目的长期目标是通过开发 基于时间分辨红外振动光谱的结构技术。 蛋白质红外编码丰富的结构信息，对裂解或 质子转移过程中X-H键的形成。时间分辨红外技术包括三个步骤： I)检测捕获蛋白质结构动力学的时间分辨红外信号；ii)识别 氨基酸对特定的红外信号有贡献；iii)可靠地将红外信号转化为质子 蛋白质中的位置。在这个项目中，我们将重点探测质子位置的动态变化 以细菌蓝光感受器(PYP)为模型的蛋白质功能中的组氨酸侧链 系统。组氨酸的三种质子化状态是His(两个质子，在N和N上)，His0D(唯一质子 在N上)和His0E(N上的唯一质子)。目标1是检测掩埋的His108和His108的pH诱导的质子化 静态PYP中暴露在溶剂中的HIS3；目标2是检测质子化状态随时间分辨的动态变化 His108在光激活时处于信号状态；目标3是检测化学激活的时间- 解决了PYP中的质子转移问题。我们将使用高精度FT-IR(目标1)，时间分辨快速扫描FT-IR (目标2)，并将微流控快速混合装置与FT-IR显微镜相结合，收集了16,000 使用焦平面阵列探测器一次傅里叶变换红外光谱(目标3)。我们将使用红外线信号分配 特定的同位素编辑与特定部位的突变相结合。组氨酸质子化状态将被推导出来 使用我们最近开发的振动结构标记带。 PI在先进的红外线技术方面有丰富的经验，是俄克拉何马州的主任 高级红外生物学中心。主要合作者是PYP、分子生物学和 PYP的同位素编辑。NSF MRI资助的最先进的FT-IR系统将用于该项目。六 本科生将进行FT-IR实验，准备各种PYP样品，进行FT-IR 红外数据分析，为FT-IR数据解释做出贡献，并在专业会议上展示结果。 将通过以下方式鼓励女性、美国原住民学生和其他少数族裔学生加入该项目 大学的三个项目1)俄克拉荷马州路易斯·斯托克斯少数族裔参与联盟，2)中心 主权国家，以及3)新生研究学者计划。

4. Project Summary/Abstract Proton-transfer in protein is a fundamental process underlying protein functions including signaling/regulation, bio-energetics, and bio-catalysis. Understanding what triggers proton-transfer and how proton-transfer drives subsequent functionally important structural transformations are of significant importance to understand the structure-function relations of many proteins. Such research is significantly hampered by the paucity of widely accessible structural techniques for probing dynamic changes in proton positions during protein function. The long-term goal of this project is to overcome this barrier by developing a time-resolved infrared vibrational spectroscopy-based structural technology. Protein infrared encode rich structural information and are particularly sensitive to cleavage or formation of X-H bonds during proton-transfer. Time-resolved infrared technology consists of three steps: i) detecting time-resolved infrared signals that capture protein structural dynamics; ii) identifying which amino acids contribute to specific infrared signals; iii) reliably translating infrared signals into proton positions in proteins. In this project, we will focus on detecting dynamic changes in proton positions in histidine side chains during protein function, using a bacterial blue-light photoreceptor (PYP) as a model system. The three protonation states of histidine are His+ (two protons, on N & N), His0D (a sole proton on N), and His0E (a sole proton on N). Aim 1 is to detect pH-induced protonation of buried His108 and solvent-exposed His3 in static PYP; Aim 2 is to detect time-resolved dynamic changes in protonation states of His108 in the signaling state upon light activation; and Aim 3 is to detect chemically activated time- resolved proton transfer in PYP. We will use high-precision FT-IR (Aim 1), time-resolved rapid-scan FT-IR (Aim 2), and integrating a microfluidic rapid-mixing device with an FT-IR microscope for collecting 16,000 FT-IR spectra at once using a focal plane array detector (Aim 3). We will assign infrared signals using specific isotope-editing combined with site-specific mutations. Histidine protonation states will be derived using vibrational structural marker bands that we recently developed. The PI has extensive experience in advanced infrared technologies and is the director of Oklahoma Center for Advanced Infrared Biology. The key collaborator is an expert in PYP, molecular biology and isotope-editing of PYP. An NSF MRI funded state-of-the-art FT-IR system will be used for this project. Six undergraduate students will carry out the FT-IR experiments, prepare various PYP samples, perform FT- IR data analysis, contribute to FT-IR data interpretation, and present results at professional conferences. Women, native American students, and other minority students will be encouraged to join the project via University’s three programs 1) Oklahoma Louis Stokes Alliance for Minority Participation, 2) the Center for Sovereign Nations, and 3) the Freshman Research Scholar program.