TRANSIENT INFRARED PROBING OF PROTEIN FOLDNG AND CONFORMATIONAL DYNAMICS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。蛋白质折叠问题被认为是结构生物学的基本问题之一。折叠研究是当今一个非常活跃的领域，在分子水平上探索折叠的实验和理论技术越来越精致。其目的是为理解和预测蛋白质折叠途径、稳定结构以及热力学和动力学上可获得的构象亚态提供实验和理论基础，给出初级氨基酸序列。对蛋白质折叠的定量理解显然对蛋白质工程很重要。了解蛋白质如何折叠也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。有人建议开发能够触发和探测蛋白质(以及其他分子系统)在不同时间尺度上的构象变化的仪器。时间分辨红外光谱(IR)为在具有结构特异性的分子水平上监测动力学事件提供了极大的灵活性和能力，并将被用来生成对感兴趣的时间范围内的瞬时物种及其动力学的详细结构解释。利用这些仪器，我们建议主要研究蛋白质是如何折叠的。计划进行一系列详细的实验，以详细了解蛋白质二级结构和三级结构的形成。我们正在进一步扩展现有的仪器，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。开发一台纳秒温度跳跃(T-JUMP)红外光谱仪的具体目标正在继续和扩展，该光谱仪既可以测量离散频率的瞬时动力学，也可以测量离散反应时间的时间分辨光谱。微秒级FTIR耦合连续流动混合和毫秒级FTIR耦合停流装置的开发现已可用。二维(2D)相关分析使特定地点的构象研究和作为动力学探针的CN运动的探索成为可能。对α-螺旋多肽的螺旋-螺旋转变以及β-发夹模型多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仪与荧光检测相结合，并将ATR光谱结合到我们的IR功能中来研究膜蛋白。该项目的另一个方向将是多肽/蛋白质聚集的研究。多肽和蛋白质聚集是许多疾病的根本原因。这个项目的目的是通过系统的方法了解多肽聚集的一些基本方面。例如，关于β-发夹的初步结果表明，β-转折在控制β-聚集体的形成方面发挥着重要作用。

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.