Developing Infrared 'FRET' Analogs to Capture Molecular Snapshots through Non-equilibrium 2D IR Spectroscopy of Recognition and Self-Assembly in Biologically Relevant Systems

项目摘要。尽管有强烈的兴趣，生物分子的三维结构及其动力学的研究 由于生长适合于X射线衍射的3D晶体的固有困难以及其 对于溶液NMR研究，溶解度差。我们提出了一个瞬态二维红外方法，将解决的问题， 构象动力学和结构变化的主链和侧链运动直接，特别是当 生物分子在明确定义的初始条件下开始，然后在短脉冲光解时， 由此产生的结构分布可以通过2D IR光谱来跟踪。在这项研究中，A 将开发光谱工具，以绘制出两种结构运动，同时提供对 每个标记位点的溶剂动力学以及它们相应的位置如何促进分子 通过弱缔合力的识别和自组装。关键结构的快速动力学 RNA或抗菌肽（AMP）作用中的事件将在时间尺度上测量， 旋转周期（fs-ps）的显着构象重组所需的（ns-ms），通过采用 我们的瞬态二维红外方法。对非平衡动力学的真实的时间观测将提供一个原子 选择的结构如何遍历反应路径到稳定的最终状态的水平视图。这些信息将 用于挑战和测试尖端的非平衡分子动力学模拟。 本文概述的研究旨在将联合收割机技术（例如：光引发、pH跳变等） 传统上用于确定线性光谱中的动力学，其信息包来自 用二维红外光谱进行探测。二维红外光谱将提供足够的结构和时间分辨率， 产生分子运动的快照，沿着特定生物事件的反应途径。特别是， 我们将同时测量生物分子内的距离和角度， 动力学，包括氢键交换，耦合水动力学和极性残留场波动，周围 每个单独的探针。通过利用各种启动技术的优势，我们将剖析侧面 链运动和整体结构变化负责分子识别，折叠和分子 AMP活性的组装。此外，我们将解开的损失氢键，基地堆叠， 进化的紧凑性，以揭示RNA折叠/解折叠的机制途径的分子细节。 更广泛的目标是获得一个化学键尺度的相互作用，导致 生产性构象变化。尽管RNA错误折叠被认为是导致自身免疫性疾病的原因， 疾病，如狼疮，他们不像蛋白质错误折叠导致阿尔茨海默氏病， example.这项工作将有助于揭示这些非天然折叠的原因。此外，关于AMP， 其中一些裂解肽可能是破坏癌细胞的关键，并标志着癌细胞的发展。 可以靶向特定脂质组合物的治疗剂。 行政设备补充说明。如上所述，母提案涉及 肽与各种膜模拟物和RNA相互作用的准或非平衡动力学 通过2D IR和瞬态2D IR非线性激光光谱测量折叠/展开。执行这些 实验中，利用相干天秤座超快Ti：Sapphire放大器（具有80 fs脉冲）来最终产生 2D IR光子回波测量所需的中红外脉冲。该设备补充是 要求购买更换Evolution 30激光二极管头。Evolution 30激光二极管头 相干天秤座飞秒激光器稳定放大所需的泵浦激光器的主要组成部分 激光系统没有有效的放大，就不可能产生稳定的飞秒脉冲 与母授权相关的所有2D IR和瞬态2D IR测量（R15 GM 1224597）。 5年后，激光二极管开始失效，导致转换效率降低，最终导致 不能适当地泵送放大器系统，最终产生不稳定性。原始激光 系统是用PI的大学启动资金购买的，而且是在5年前。因此，由于一些 最近的整体效率下降，并与相干激光技术人员进行了讨论，一致认为， 激光二极管头的使用寿命已接近尾声，安装新组件的成本为46，804美元 是杰出的因此，基于这一理由，我要求提供上述设备补充。

Project Summary. Despite strong interest, the study of the 3D structures of biomolecules and their dynamics remain challenging by the inherent difficulty in growing 3D crystals suitable for X-ray diffraction and by their poor solubility for solution NMR studies. We propose a transient 2D IR approach that will address questions of conformational dynamics and structural change of backbone and side chain motions directly, especially when the biomolecule begins in a well-defined initial condition, and then upon short pulse photolysis, evolution of the resulting structure distributions can be tracked by 2D IR spectroscopy. In the course of this research, a spectroscopic tool will be developed to map out both structural motions while concurrently providing insight into the solvent dynamics at each labelled site and how their corresponding locations promote the molecular recognition and self-assembly through weak associative forces. The fast dynamics during the key structural events in RNA or antimicrobial peptide (AMP) action will be measured on time scales ranging from single bond rotational periods (fs-ps) to those required for significant conformational reorganization (ns-ms) by employing our transient 2D IR methods. Observations in real time of the non-equilibirum dynamics will provide an atomic level view of how chosen structures traverse reaction paths to stable final states. This information will then be used to challenge and test cutting edge non-equilibrium molecular dynamics simulations. The research outlined herein aims to combine techniques (eg. photo-initation, pH-jump, etc.) traditionally used to determine kinetics in linear spectroscopies with the information package that comes from probing with 2D IR spectroscopy. 2D IR spectroscopy will afford sufficient structural and time resolution to generate snapshots of molecular motions along the reaction pathway of specific biological events. In particular, we will simultaneously measure distances and angles within biomolecules and also detect the local vibrational dynamics, including H-bond exchange, coupled water dynamics and polar residue field fluctuations, around each individual probe. By harnessing the strengths of various initiation techniques, we will dissect the side chain motions and global structural changes responsible for molecular recognition, folding, and molecular assembly of AMP activity. Furthermore, we will disentangle the loss of hydrogen bonding, base stacking, and evolving compactness to uncover molecular details of the mechanistic pathway of RNA folding/unfolding. The broader objective is to obtain a chemical bond scale description of interactions that lead to productive conformational changes. Although RNA misfolds are believed to be responsible for autoimmune diseases such as lupus, they are not as well understood as protein misfolds leading to Alzheimer's disease for example. This work will help uncover the reasons for these non-native folds. Moreover, in regards to AMPs, some of these lytic peptides may hold the key to destroy cancer cells and mark the way for the development of therapeutics that can target specific lipid composition. Administrative Equipment Supplement Justification. As mentioned above, the parent proposal concerns quasi- or non-equilibrium dynamics of the interactions of peptides with various membrane mimics and RNA folding/unfolding measured via 2D IR and transient 2D IR nonlinear laser spectroscopy. To perform these experiments, a Coherent Libra ultrafast Ti:Sapphire amplifier (with 80fs pulses) is utilized to ultimately generate the mid-IR pulses necessary for the 2D IR photon echo measurements. This equipment supplement is requested for purchasing a replacement Evolution 30 laser diode head. The Evolution 30 laser diode head is the major component of the pump laser required for stable amplification of the Coherent Libra femtosecond laser system. Without efficient amplification, it is impossible to generate the stable femtosecond pulses necessary for all 2D IR and transient 2D IR measurements relevant to the parent grant (R15GM1224597). After 5 years, the laser diodes start to fail resulting in a reduction of conversion efficiency eventually leading to the inability to pump the amplifier system appropriately, ultimately creating instabilities. The original laser system was purchased with the PI's university startup funds and it was exactly 5 years ago. Thus, due to some recent drops in overall efficiency and a discussion with the Coherent laser technician, it was agreed that the laser diode heads are close to the end of their lifetime and a new assembly with installation costing $46,804 was eminent. So, it is with this justification that I am requesting the aforementioned equipment supplement.