Advanced Infrared Biology of Protein Structure & Dynamics

蛋白质结构的高级红外生物学

• 批准号: 10360289
• 负责人: AIHUA XIE
• 金额: $ 43.8万
• 依托单位: OKLAHOMA STATE UNIVERSITY STILLWATER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10360289
• 关键词: Active Sites; Amides; Amino Acids; Ants; Binding; Bioenergetics; Biological Models; Biology; Catalysis; Charge; Chemicals; Data Analyses; Detection; Devices; Enzymes; Funding; Goals; Guanosine Triphosphate; Histidine; Hydrogen Bonding; Hydrophobicity; Imidazole; Isotopes; Lasers; Left; Ligands; Light; Location; Magnetic Resonance Imaging; Malignant Neoplasms; Methods; Microfluidic Microchips; Microfluidics; Microscope; Minority Participation; Molecular Biology; Molecular Conformation; Mutation; NCI Scholars Program; Native Americans; Oklahoma; Pharmaceutical Preparations; Photoreceptors; Positioning Attribute; Process; Protein Conformation; Protein Dynamics; Proteins; Protons; Pump; Receptor Activation; Regulation; Research; Rest; STEM field; Sampling; Scanning; Side; Signal Transduction; Signaling Protein; Site; Site-Directed Mutagenesis; Solvents; Spectrum Analysis; Stimulus; Structure; Students; System; Techniques; Technology; Tertiary Protein Structure; Testing; Time; Translating; Universities; Woman; base; detector; drug discovery; experience; experimental study; human disease; imaging detector; innovation; minority student; nanosecond; photoactivation; programs; protein function; protein structure; protein structure function; protonation; prototype; receptor; symposium; time use; tool; undergraduate student; vibration

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.

4.项目总结/摘要 蛋白质中的质子转移是蛋白质功能的基本过程， 信号/调节、生物能量学和生物催化。了解什么触发质子转移， 质子转移如何驱动随后的功能重要的结构转变是重要的 这对理解许多蛋白质的结构-功能关系非常重要。这样的研究意义重大。 由于缺乏广泛使用的结构技术来探测质子的动态变化， 在蛋白质功能中的位置。该项目的长期目标是通过开发 一种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，并对切割或 在质子转移过程中形成X-H键。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; 氨基酸有助于特定的红外信号; iii）可靠地将红外信号转化为质子 蛋白质的位置。在这个项目中，我们将重点探测质子位置的动态变化， 组氨酸侧链在蛋白质功能，使用细菌蓝光感光器（PYP）作为模型 系统组氨酸的三种质子化状态是His+（两个质子，在N+和N+上）、His 0 D（唯一质子 和His 0 E（NH3上的唯一质子）。目的1是检测pH诱导的掩埋的His 108的质子化， 静态PYP中溶剂暴露的His 3;目标2是检测质子化状态的时间分辨动态变化 目的3是检测光活化后His 108在信号传导状态下的化学活化时间。 解决了PYP中的质子转移。我们将使用高精度FT-IR（目标1），时间分辨快速扫描FT-IR (Aim 2）将微流控快速混合装置与FT-IR显微镜集成， 使用焦平面阵列检测器立即获得FT-IR光谱（目标3）。我们将分配红外信号使用 特异性同位素编辑结合位点特异性突变。组氨酸质子化状态将被导出 使用我们最近开发的振动结构标记带。 PI在先进的红外技术方面拥有丰富的经验，是俄克拉荷马州的主管 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，制备各种PYP样品，进行FT- 红外数据分析，有助于FT-IR数据解释，并在专业会议上展示结果。 妇女，美国土著学生和其他少数民族学生将被鼓励参加该项目，通过 大学的三个项目1）俄克拉荷马州路易斯斯托克斯少数民族参与联盟，2）中心 主权国家，和3）新生研究学者计划。