TRANSIENT INFRARED PROBING OF PROTEIN FOLDNG AND CONFORMATIONAL DYNAMICS

蛋白质折叠和构象动力学的瞬态红外探测

• 批准号: 7373126
• 负责人: FENG GAI
• 金额: $ 6.77万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7373126
• 关键词: TRANSIENT; INFRARED; PROBING; PROTEIN; FOLDNG

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The protein folding problem is considered to be one of the fundamental questions in structural biology. Folding research is today a very active area, where the experimental and theoretical techniques for probing folding at the molecular level are becoming more and more refined. The goal is to provide an experimental and theoretical basis for understanding and predicting protein folding pathways, the stable structures, and thermally and kinetically accessible conformation substates, given the primary amino acid sequence. A quantitative understanding of protein folding is apparently important for protein engineering. Understanding how proteins fold can also help to interpret quantitatively the structure-function relationships and folding related diseases. Furthermore, a predictive understanding of protein folding will accelerate the discovery of information contained in the large number of gene sequences that are now becoming available. It was proposed to develop instruments that are capable of triggering and probing conformational changes in proteins (and other molecular systems as well) on various timescales. Time-resolved infrared (IR) spectroscopy offers great flexibility and power for monitoring kinetic events on the molecular level with structure specificity and will be used to generate detailed structure interpretations of the transient species and their dynamics over the time range of interest. Using these instruments, we propose to study primarily how proteins fold. A detailed set of experiments are planned to gain detailed insight into the formation of protein secondary and tertiary structures. We are further extending current instruments and developing new instruments that are capable of triggering and probing conformational changes in proteins on various timescales. The specific aim of developing a nanosecond temperature-jump (T-jump) infrared spectrometer that can measure both transient kinetics at discrete frequencies and time-resolved spectra at discrete reaction times is being continued and extended. The microsecond FTIR coupled continuous-flow mixing and the millisecond FTIR coupled stopped-flow apparatus development is now available. The 2-dimensional (2D) correlation analysis has permitted site specific conformation studies and explorations of CN motions as a probe of dynamics. Studies of the helix-coil transition in alpha-helical peptides were performed, as well as studies of the stability and folding kinetics of beta-hairpin model peptides. We further work on the combination of the stop-flow apparatus with fluorescence detection and the incorporation of ATR spectroscopy into our IR capabilites to study membrane proteins. Another direction of this project will be the study of peptide/protein aggregation. Peptide and protein aggregation is the underlying cause of many diseases. This project is aimed to understand some fundamental aspects of peptide aggregation through a systematic approach. For example preliminary results on beta-hairpins suggest that the beta-turn plays a significant role in controlling the formation of beta-aggregates.

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中出现。列出的机构是中心的机构，不一定是研究者的机构。蛋白质折叠问题被认为是结构生物学的基本问题之一。折叠研究如今是一个非常活跃的领域，在分子水平上探测折叠的实验和理论技术变得越来越完善。目标是在给定一级氨基酸序列的情况下，为理解和预测蛋白质折叠途径、稳定结构以及热和动力学可接近的构象亚状态提供实验和理论基础。对蛋白质折叠的定量理解对于蛋白质工程显然很重要。了解蛋白质如何折叠还有助于定量解释结构-功能关系和折叠相关疾病。此外，对蛋白质折叠的预测性理解将加速发现目前可用的大量基因序列中包含的信息。有人建议开发能够在不同时间尺度上触发和探测蛋白质（以及其他分子系统）构象变化的仪器。时间分辨红外 (IR) 光谱为监测具有结构特异性的分子水平上的动力学事件提供了极大的灵活性和功能，并将用于生成瞬态物质及其在感兴趣的时间范围内的动态的详细结构解释。使用这些仪器，我们建议主要研究蛋白质如何折叠。计划进行一组详细的实验，以详细了解蛋白质二级和三级结构的形成。我们正在进一步扩展现有仪器并开发能够在不同时间尺度上触发和探测蛋白质构象变化的新仪器。开发纳秒温跃（T-jump）红外光谱仪的具体目标正在继续和扩展，该光谱仪可以测量离散频率下的瞬态动力学和离散反应时间下的时间分辨光谱。微秒 FTIR 耦合连续流混合和毫秒 FTIR 耦合停流装置现已开发。二维 (2D) 相关性分析允许对 CN 运动进行位点特定构象研究和探索作为动力学探针。对α-螺旋肽中的螺旋-螺旋转变进行了研究，并对β-发夹模型肽的稳定性和折叠动力学进行了研究。我们进一步致力于将停流装置与荧光检测相结合，并将 ATR 光谱纳入我们的红外功能中，以研究膜蛋白。该项目的另一个方向是肽/蛋白质聚集的研究。肽和蛋白质聚集是许多疾病的根本原因。该项目旨在通过系统方法了解肽聚集的一些基本方面。例如，β-发夹的初步结果表明，β-转角在控制β-聚集体的形成中发挥着重要作用。