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

开发红外“FRET”类似物，通过生物相关系统中的非平衡二维红外光谱识别和自组装捕获分子快照

基本信息

批准号：
9730143
负责人：
Matthew J Tucker
金额：
$ 4.68万
依托单位：
UNIVERSITY OF NEVADA RENO
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-08-01 至 2020-07-31
项目状态：
已结题

项目摘要

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.

项目摘要。尽管人们对生物分子的 3D 结构及其动力学的研究抱有浓厚的兴趣由于生长适合 X 射线衍射的 3D 晶体的固有困难及其溶液 NMR 研究的溶解度较差。我们提出了一种瞬态 2D IR 方法，该方法将解决以下问题直接主链和侧链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，然后通过短脉冲光解作用，进化出由此产生的结构分布可以通过二维红外光谱进行跟踪。在本研究过程中，一个将开发光谱工具来绘制两种结构运动，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联合力进行识别和自组装。关键结构过程中的快速动态 RNA 或抗菌肽 (AMP) 作用中的事件将在单键范围内的时间尺度上进行测量通过使用旋转周期（fs-ps）到显着构象重组（ns-ms）所需的周期我们的瞬态二维红外方法。对非平衡动力学的实时观察将提供原子所选结构如何穿越反应路径达到稳定最终状态的水平视图。该信息随后将被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技术（例如光引发、pH 跳跃等）与传统上用于确定线性光谱中的动力学，其信息包来自 2D IR 光谱探测。二维红外光谱将提供足够的结构和时间分辨率沿着特定生物事件的反应途径生成分子运动的快照。尤其，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、耦合水动力学和极性残留场波动，每个单独的探头。通过利用各种启动技术的优势，我们将剖析侧面链运动和全局结构变化负责分子识别、折叠和分子 AMP 活性的组装。此外，我们将解开氢键的损失、碱基堆积和不断发展的紧凑性以揭示 RNA 折叠/解折叠机制途径的分子细节。更广泛的目标是获得相互作用的化学键尺度描述，从而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对于狼疮等疾病，人们对它们的了解不如对导致阿尔茨海默氏病的蛋白质错误折叠了解的多。例子。这项工作将有助于揭示这些非原生折叠的原因。此外，关于 AMP，其中一些裂解肽可能是摧毁癌细胞的关键，并为癌症的发展指明了道路。可以针对特定脂质成分的疗法。行政设备补充理由。如上所述，家长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。为了执行这些实验中，利用 Coherent Libra 超快钛宝石放大器（具有 80fs 脉冲）最终生成二维红外光子回波测量所需的中红外脉冲。这个装备补充是请求购买替换 Evolution 30 激光二极管头。 Evolution 30 激光二极管头是相干 Libra 飞秒稳定放大所需的泵浦激光器的主要组件激光系统。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 (R15GM1224597) 相关的所有 2D IR 和瞬态 2D IR 测量是必需的。 5 年后，激光二极管开始失效，导致转换效率降低，最终导致无法适当地泵送放大器系统，最终造成不稳定。原来的激光系统是用PI的大学启动资金购买的，距今整整5年了。因此，由于一些最近整体效率下降，并与相干激光技术人员进行讨论，一致认为激光二极管头已接近其使用寿命，新组件的安装成本为 46,804 美元是杰出的。因此，我正是基于这个理由，要求上述设备的补充。