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

• 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
• 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
• 来源: https://reporter.nih.gov/project-details/9730143
• 关键词: Address; Alzheimer' s Disease; Amplifiers; Autoimmune Diseases; Biological; Coupled; Crystallization; Drops; Equipment; Event; Evolution; Fluorescence Resonance Energy Transfer; Funding; Head; Hydrogen Bonding; Individual; Kinetics; Label; Lasers; Lead; Lipids; Location; Lupus; Lytic; Maps; Measurement; Measures; Membrane; Methods; Molecular; Molecular Conformation; Motion; Parents; Pathway interactions; Peptides; Photons; Physiologic pulse; Proteins; Pump; RNA; RNA Folding; Reaction; Research; Resolution; Rotation; Sapphire; Side; Site; Solubility; Solvents; Spectrum Analysis; Structure; System; Techniques; Testing; Time; Universities; Vertebral column; Water; Work; X ray diffraction analysis; analog; antimicrobial peptide; base; cancer cell; chemical bond; cost; experimental study; insight; interest; molecular assembly/self assembly; molecular dynamics; molecular recognition; non-Native; parent grant; photolysis; self assembly; single bond; spectroscopic survey; therapeutic development; therapeutic target; three dimensional structure; tool; vibration

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。尽管有浓厚的兴趣，但对生物分子的3D结构及其动力学的研究 由于固有的困难在生长适合X射线衍射的3D晶体方面保持挑战 溶液NMR研究的溶解度差。我们提出了一种瞬态2D IR方法，该方法将解决 直接直接直接 生物分子以明确定义的初始条件开始，然后在短脉冲光解时开始 可以通过2D红外光谱跟踪产生的结构分布。在这项研究过程中， 光谱工具将被开发以绘制这两个结构运动，同时提供洞察力 每个标记位点的溶剂动力学以及它们相应位置如何促进分子 通过微弱的联合力量的认可和自组装。关键结构期间的快速动态 RNA或抗菌肽（AMP）作用的事件将以单键范围的时间尺度进行测量 旋转周期（FS-PS）到通过采用显着构象重组（NS-MS）所需的旋转周期（FS-PS） 我们的瞬态2D IR方法。实时观察非等分临床动力学将提供原子 所选结构如何穿越稳定最终状态的反应路径的水平视图。然后，此信息将是 用于挑战和测试尖端非平衡分子动力学模拟。 本文概述的研究旨在结合技术（例如，摄影，pH-Jump等） 传统上用于确定线性光谱中的动力学，并带有来自 用2D IR光谱探测。 2D红外光谱法可以提供足够的结构和时间分辨率 沿特定生物学事件的反应途径产生分子运动的快照。尤其， 我们将同时测量生物分子内的距离和角度，并检测到局部振动 动力学，包括H键交换，耦合的水动力学和极地残留场波动，周围 每个探测器。通过利用各种启动技术的优势，我们将剖析侧面 链运动和全球结构变化负责分子识别，折叠和分子 AMP活动组装。此外，我们将解散氢键，基础堆叠和 不断发展的紧凑性，以发现RNA折叠/展开机械途径的分子细节。 更广泛的目标是获得相互作用的化学键尺度描述，导致 生产构象变化。尽管据信RNA错误造成自身免疫性 狼疮等疾病不像导致阿尔茨海默氏病的蛋白质折叠术那样充分理解 例子。这项工作将有助于发现这些非本地折叠的原因。此外，关于放大器， 这些裂解肽中的某些可能持有破坏癌细胞的关键，并为开发的发展标志 可以靶向特定脂质组成的治疗剂。 行政设备补充辩护。如上所述，父母建议涉及 肽与各种膜模仿和RNA相互作用的准或非平衡动力学 通过2D IR和瞬态2D IR非线性激光光谱法测量的折叠/展开。执行这些 实验，一种连贯的天秤座超快Ti：蓝宝石放大器（具有80fs脉冲）最终生成 2D IR光子回波测量所需的MID-IR脉冲。该设备补充是 要求购买替换进化30激光二极管头。进化30激光二极管头是 稳定放大的泵激光器的主要组成部分是稳定的。 激光系统。没有有效的放大，就无法产生稳定的飞秒脉冲 对于所有与父授予赠款相关的2D IR和瞬态2D IR测量所必需的（R15GM1224597）。 5年后，激光二极管开始失败，导致转化效率的降低最终导致 无法适当地泵送放大器系统，最终创造不稳定性。原始激光 系统是通过PI的大学初创企业基金购买的，正好是5年前。因此，由于一些 最近的总体效率下降以及与连贯的激光技术员的讨论，同意 激光二极管头靠近他们一生的末期，安装的新组装售价为46,804美元 是杰出的。因此，正是通过这种理由，我要求上述设备补充。

项目成果

Synthesis of 5-Cyano-Tryptophan as a Two-Dimensional Infrared Spectroscopic Reporter of Structure.

• DOI: 10.1002/anie.201803849
• 发表时间: 2018-06-18
• 期刊: Angewandte Chemie (International ed. in English)
• 作者: Chalyavi F;Gilmartin PH;Schmitz AJ;Fennie MW;Tucker MJ
• 通讯作者: Tucker MJ

Nanostructured Ni-Cu Electrocatalysts for the Oxygen Evolution Reaction.

• DOI: 10.1039/d0cy00427h
• 发表时间: 2020-08-07
• 期刊: Catalysis science & technology
• 影响因子: 5
• 作者: Gautam RP;Pan H;Chalyavi F;Tucker MJ;Barile CJ
• 通讯作者: Barile CJ

Interspecies Bombolitins Exhibit Structural Diversity upon Membrane Binding, Leading to Cell Specificity.

• DOI: 10.1016/j.bpj.2019.02.005
• 发表时间: 2019-03
• 期刊: Biophysical journal
• 影响因子: 3.4
• 作者: Matthew G Roberson;Devin K. Smith;S. White;I. Wallace;M. J. Tucker