2D IR DUAL FREQUENCY AND DUAL ISOTOPE REPLACEMENT STRATEGIES

2D IR 双频和双同位素替代策略

• 批准号: 8169536
• 负责人: ROBIN Main HOCHSTRASSER
• 金额: $ 16.62万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8169536
• 关键词: Address; Amides; Area; Binding; Biological; Biological Models; Carbon; Cell Surface Receptors; Cell physiology; Computer Retrieval of Information on Scientific Projects Database; Coupled; Coupling; Development; Dimerization; Dipeptides; Engineering; Ensure; Environment; Equilibrium; Frequencies; Funding; Future; Glycophorin A; Goals; Grant; Hydrogen Bonding; Individual; Institution; Investigation; Ion Channel; Isotopes; Joints; Lasers; Membrane; Membrane Proteins; Methods; Micelles; Molecular Conformation; N-methylacetamide; Optics; Oxidation-Reduction; Peptides; Physiologic pulse; Process; Proteins; Protocols documentation; Pump; Reporting; Research; Research Personnel; Resources; Side; Signal Transduction; Solubility; Solutions; Source; Spectrum Analysis; Stretching; System; Technology; Transmembrane Domain; United States National Institutes of Health; Vertebral column; Vesicle; Water; Width; Work; X ray diffraction analysis; X-Ray Diffraction; base; cost; density; dimer; driving force; infancy; interest; peptide structure; prototype; research study; theories; three dimensional structure; Address; Amides; Area; Binding; Biological; Biological Models; Carbon; Cell Surface Receptors; Cell physiology; Computer Retrieval of Information on Scientific Projects Database; Coupled; Coupling; Development; Dimerization; Dipeptides; Engineering; Ensure; Environment; Equilibrium; Frequencies; Funding; Future; Glycophorin A; Goals; Grant; Hydrogen Bonding; Individual; Institution; Investigation; Ion Channel; Isotopes; Joints; Lasers; Membrane; Membrane Proteins; Methods; Micelles; Molecular Conformation; N-methylacetamide; Optics; Oxidation-Reduction; Peptides; Physiologic pulse; Process; Proteins; Protocols documentation; Pump; Reporting; Research; Research Personnel; Resources; Side; Signal Transduction; Solubility; Solutions; Source; Spectrum Analysis; Stretching; System; Technology; Transmembrane Domain; United States National Institutes of Health; Vertebral column; Vesicle; Water; Width; Work; X ray diffraction analysis; X-Ray Diffraction; base; cost; density; dimer; driving force; infancy; interest; peptide structure; prototype; research study; theories; three dimensional structure

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 2D IR spectra of peptide modes in a variety of environments have now been examined by means of dual frequency 2D IR. In this method the two modes of interest are both incorporated into the same nonlinear signal so their joint signal exists only when they are coupled in some manner. Another approach that we have introduced is the dual isotope replacement which is a strategy for exposing structural proximities by means of 2D IR. The successful preliminary results using these methods have prompted more ambitious experiments that can answer new types of questions. Dual frequency methods are needed because the bandwidth of infrared laser pulses are too narrow to simultaneously access widely separated vibrational modes. In terms of dual frequency experiments a sufficient number of examples have been reported to make it clear that the approach has great potential but that the method is in its infancy. Only a few frequencies have been incorporated into the 2D IR experiment and mainly the strong peptide backbone modes have been accessed. The first dual frequency results with the pump/probe 2D IR method showed beautifully the coupling between the N-H and the C=O groups of dipeptides and N-methylacetamide. Results were also reported for heterodyned signals arising from peptides interacting with two frequencies. The method was applied to dipeptides and most recently to model systems that dramatized the amplification of the signal expected when weak transitions are coupled to strong ones. These experiments provide the opportunity to probe details of peptide structure and dynamics that cannot easily be accessed by conventional approaches. Not only can the individual amide modes covering a wide range of frequencies be accessed but engineered probes such as those that contain CN groups in a transparency region of water could deliver a new set of structural constraints. In another example, selective deuteration of carbon hydrogen bonds can expose C-D bonds for 2D IR dual frequency analysis as discussed in the next paragraph. It is important to develop 2D IR methods for the study of membrane proteins, which are vital components of the cell physiology and include the alpha-helical class of cell-surface receptors, ion channels, transporters and redox proteins. Many have a single transmembrane (TM) helix that associates with other TM helices to form helical bundles. These assemblies occur a variety of biological situations and also have advantages for the study of folding in membranes. Despite the strong interest in them, study of their 3D structures and their dynamics remains challenging by the inherent difficulty in growing 3D crystals suitable for X-ray diffraction and by their poor solubility for solution NMR studies. The TM domain of glycophorin A (GpA) helical dimers present a prototype system for 2D IR to address the structural basis of helix association. This domain is indicated to be responsible for protein dimerization and only a few residues compose the dimerization interface. 2D IR methods will also be configured to access features that stabilize the folded conformations of membrane proteins. In the folding of helical membrane proteins the driving forces might be dispersion force interactions and/or the strong hydrogen bonds formed in the membrane. With water-soluble proteins there are energetic costs of changing a buried non-polar side chain to a smaller side chain. Understanding of the folds of membrane proteins in micelles is just beginning to emerge. Work in this area will provide a particularly fertile avenue for future investigations using 2D IR methods on isotopically edited transmembrane helices that expose both the equilibrium dynamics and the structural arrangements of coupled residues in terms of their spatial arrangements across the membrane. Interhelical H-bonds are also important in the stabilization of helix-helix interactions and 2D IR is now known to be sensitive to interactions across hydrogen bonds Current goals within this Core project are: - Completion of 50 fs dual optical parametric oscillators to access frequencies from the O-H and N-H stretches region down to the amide-III at ca. 10 for dual frequency 2D IR. A large band width in each of the pulses will ensure that cross peak spectra can be recorded over the widest possible frequency range and that the joint correlations of the two modes can be clearly identified. - Development of dual frequency technologies for recording of proximities and couplings by 2D IR between amide-I, C-D, N-H, O-H and amide modes in soluble and trans-membrane peptides in vesicles, micelles and bicelles. - Theory and processing of the 2D IR spectra of dual isotopic edited peptides and multiple isotopomers of peptide aggregates. - Introduction of high optical density protocols to dual frequency 2D IR spectroscopy permitting the study of the weak C¿H mode coupling to strong amide modes in membrane bound helix dimers.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 现在已经通过双频率2D IR检查了各种环境中的辣椒模式的2D红外光谱。在这种方法中，两种感兴趣的模式都被掺入相同的非线性信号中，因此仅在以某种方式耦合时才存在它们的关节信号。我们引入的另一种方法是双同位素置换，这是通过2D IR揭示结构接近的策略。使用这些方法成功的初步结果促使了更雄心勃勃的实验，可以回答新的问题。需要双重频率方法，因为红外激光脉冲的带宽太窄，无法简单地访问广泛分开的振动模式。就双重频率实验而言，据报道，已经有足够数量的示例表明该方法具有很大的潜力，但该方法仍处于起步阶段。仅将少数频率纳入了2D IR实验中，并且主要访问了强肽骨架模式。泵/探针2D IR方法的第一个双频频率显示了N-H和C = O组之间的耦合和N-甲基乙酰氨酰胺之间的耦合。还报道了由于辣椒与两个频率相互作用的辣椒引起的异质信号的结果。该方法应用于二肽，最近一次应用于模型系统，这些系统戏剧化了当弱转变与强型转换耦合时预期的信号的扩增。这些实验为探测肽结构和动力学细节的细节提供了机会，这些细节无法通过常规方法轻松访问。不仅可以访问涵盖广泛频率的单个酰胺模式，而且可以进行工程问题，例如在透明的水区域中包含CN组的问题，可以提供一系列新的结构约束。在另一个示例中，如下一段中讨论的那样，碳氢键的选择性暗质键可以暴露于2D IR双重频率分析的C-D键。 为研究膜蛋白的研究开发2D IR方法很重要，膜蛋白是细胞生理的重要成分，包括细胞表面受体，离子通道，转运蛋白和氧化还原蛋白的α-螺旋类别。许多人具有与其他TM螺旋相关的单个跨膜（TM）螺旋，形成螺旋束。这些组件发生了多种生物学情况，并且在膜中折叠的研究也具有优势。尽管对它们产生了浓厚的兴趣，但对其3D结构的研究和动态的研究仍然受到继承难以生长适合X射线衍射的3D晶体以及对解决方案NMR研究的不良解决方案的挑战。糖果蛋白A（GPA）螺旋二聚体的TM结构域为2D IR提供了一个原型系统，以解决螺旋结合的结构基础。该结构域被指出是蛋白质二聚化的原因，只有少数残基二聚化界面。 2D IR方法还将配置为访问稳定膜蛋白折叠构象的功能。在螺旋膜蛋白的折叠中，驱动力可能是分散力相互作用和/或膜中形成的强氢键。使用水溶性蛋白质，将内置的非极性侧链更改为较小的侧链的有力成本。了解胶束中膜蛋白的褶皱刚刚开始出现。该领域的工作将为未来的途径提供特别肥沃的途径，使用2D IR方法在同位素编辑的跨膜螺旋上进行，以揭示其在整个膜上的空间布置方面，均揭示了耦合残基的结构排列。螺旋间H键在稳定螺旋 - 螺旋相互作用中也很重要，现在已知2D IR对跨氢键的相互作用敏感 该核心项目中的当前目标是： - 完成50 fs双光学参数振荡器，从O-H和N-H伸展区域访问频率到CA的酰胺III。 10用于双频率2D IR。每个脉冲中的较大带宽将确保可以在最宽的频率范围内记录跨峰光谱，并且可以清楚地识别两种模式的关节相关性。 - 开发双频率技术，用于在蔬菜，胶束和双丝中，在固体和跨膜肽中，酰胺I，C-D，N-H，O-H和酰胺模式之间的2D IR记录近似和耦合。 - 双同位素编辑的petides和多个同位素骨料的二维红外光谱的理论和处理。 - 将高光密度方案引入双频率2D红外光谱允许研究弱c。h模式耦合到膜结合螺旋二聚体中强酰胺模式。