TRANSIENT INFRARED PROBING OF PROTEIN FOLDNG AND CONFORMATIONAL DYNAMICS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，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完善。目标是为理解和预测蛋白质折叠途径、稳定结构以及已知初级氨基酸序列的热动力学可达构象亚态提供实验和理论基础。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包含在现在可用的大量基因序列中的信息。建议开发能够在各种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）光谱为分子水平上具有结构特异性的动力学事件监测提供了极大的灵活性和能力，并将用于生成瞬态物种的详细结构解释及其在感兴趣的时间范围内的动力学。利用这些仪器，我们打算主要研究蛋白质如何折叠。计划进行一系列详细的实验，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的仪器，并开发能够在各种时间尺度上触发和探测蛋白质构象变化的新仪器。开发纳秒温度跳变红外光谱仪的具体目标是继续和扩展，该光谱仪可以测量离散频率的瞬态动力学和离散反应时间的时间分辨光谱。目前已研制出微秒级FTIR耦合连续流混合装置和毫秒级FTIR耦合停流装置。二维（2D）相关分析允许特定位点的构象研究和探索CN运动作为动力学探针。研究了α -螺旋肽的螺旋-线圈转变，以及β -发夹模型肽的稳定性和折叠动力学。我们进一步将停止流动装置与荧光检测相结合，并将ATR光谱纳入我们的红外能力，以研究膜蛋白。该项目的另一个方向将是肽/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。该项目旨在通过系统的方法了解肽聚集的一些基本方面。例如，对β -发夹的初步结果表明，β -转在控制β -聚集体的形成中起着重要作用。

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.