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。尽管有浓厚的兴趣，但生物分子的三维结构及其动力学的研究 仍然具有挑战性，因为生长适合于X射线衍射的3D晶体的固有困难以及它们的 对于溶液核磁共振研究来说，溶解性很差。我们提出了一种瞬时2D IR方法，该方法将解决以下问题 主链和侧链直接运动的构象动力学和结构变化，特别是当 生物分子开始于明确的初始条件，然后在短脉冲光解后，生物分子的进化 由此得到的结构分布可以通过二维红外光谱来跟踪。在这项研究的过程中，一个 将开发光谱工具来绘制这两个结构运动的地图，同时提供对 每个标记位置的溶剂动力学及其相应位置如何促进分子 通过弱联想力量进行识别和自我组装。关键结构过程中的快速动力学 RNA或抗菌肽(AMP)作用的事件将在单键范围内的时间尺度上进行测量 旋转周期(f-ps)到显著构象重组所需的周期(ns-ms) 我们的瞬时2D IR方法。对非平衡动力学的实时观察将提供一个原子 所选结构如何通过反应路径到达稳定的最终状态的水平视图。然后，这些信息将被 用于挑战和测试前沿的非平衡分子动力学模拟。 这里概述的研究旨在结合技术(例如，光激发、pH跳跃等。) 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 生成沿特定生物事件的反应路径的分子运动快照。特别是， 我们将同时测量生物分子内的距离和角度，并检测局部振动 动力学，包括氢键交换、耦合水动力学和极残基场涨落 每个单独的探测器。通过利用各种引爆技术的优势，我们将剖析侧面 链运动和全球结构变化导致分子识别、折叠和分子 AMP活性的组装。此外，我们将解决氢键的损失、碱基堆叠和 进化的致密性，以揭示RNA折叠/展开的机制路径的分子细节。 更广泛的目标是获得导致以下因素的相互作用的化学键尺度描述 有成效的构象变化。尽管RNA错误折叠被认为是导致自身免疫的原因 像狼疮这样的疾病，人们对它们的理解不如蛋白质错误折叠导致阿尔茨海默病 举个例子。这项工作将有助于揭示这些非自然褶皱的原因。此外，关于AMP， 其中一些裂解肽可能掌握着摧毁癌细胞的关键，并标志着发展的道路 可针对特定脂质成分的治疗学。 行政设备补充理由。如上所述，父提案涉及 多肽与不同膜模拟物和RNA相互作用的准平衡或非平衡动力学 通过2D IR和瞬时2D IR非线性激光光谱测量折叠/展开。要执行这些操作 实验中，利用相干Libra超快钛宝石放大器(脉冲为80fs)最终产生了 二维红外光子回波测量所需的中红外脉冲。本设备补充件为 要求购买替换的Evolution 30激光二极管磁头。Evolution 30激光二极管磁头是 相干天秤座飞秒稳定放大所需的泵浦激光的主要成分 激光系统。如果没有有效的放大，就不可能产生稳定的飞秒脉冲 对于与父授权相关的所有2D IR和瞬时2D IR测量是必要的(R15GM1224597)。 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，最终造成不稳定。最早的激光器 该系统是用PI的大学启动资金购买的，而且恰好是5年前。因此，由于一些 最近总体效率下降，并与相干激光技术人员讨论后，一致认为 激光二极管磁头的寿命即将结束，安装成本为46,804美元的新组件 是杰出的。因此，正是基于这一理由，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.