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

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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 在各种环境中的肽模式的2D IR光谱现在已经检查通过双频2D IR。在这种方法中，两种模式的兴趣都被纳入到同一个非线性信号，所以它们的联合信号存在时，才以某种方式耦合。我们介绍的另一种方法是双同位素置换，这是一种通过二维红外来揭示结构邻近性的策略。使用这些方法的成功初步结果促使了更雄心勃勃的实验，可以回答新类型的问题。双频方法是必要的，因为红外激光脉冲的带宽太窄，同时访问广泛分离的振动模式。在双频实验方面，已经报道了足够数量的例子，以清楚地表明该方法具有很大的潜力，但该方法仍处于起步阶段。只有少数频率已被纳入2D IR实验，主要是强肽骨架模式已被访问。第一个双频结果与泵/探针二维红外方法显示漂亮的偶联之间的N-H和C=O基团的二肽和N-甲基乙酰胺。结果也报道了由肽与两个频率相互作用产生的外差信号。该方法被应用于二肽和最近的模型系统，戏剧化的放大信号时，预期弱转换耦合到强的。这些实验提供了机会，以探测肽的结构和动力学的细节，不能很容易地通过常规方法访问。不仅可以访问覆盖广泛频率范围的单个酰胺模式，而且工程探针（例如在水的透明区域中含有CN基团的探针）可以提供一组新的结构约束。在另一个实例中，碳氢键的选择性氘化可以暴露C-D键用于2D IR双频分析，如在下一段中讨论的。 膜蛋白是细胞生理学的重要组成部分，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。许多具有单个跨膜（TM）螺旋，其与其它TM螺旋结合以形成螺旋束。这些组件发生在各种生物学情况下，也有优势，在膜折叠的研究。尽管对它们有强烈的兴趣，但它们的3D结构及其动力学的研究仍然具有挑战性，因为生长适合X射线衍射的3D晶体的固有困难以及它们在溶液NMR研究中的溶解度差。血型糖蛋白A（GpA）螺旋二聚体的TM结构域提供了二维IR的原型系统，以解决螺旋缔合的结构基础。该结构域被指示负责蛋白质二聚化，并且只有少数残基构成二聚化界面。 2D IR方法还将被配置为访问稳定膜蛋白折叠构象的特征。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。对于水溶性蛋白质，将埋藏的非极性侧链改变为较小的侧链需要能量成本。对胶束中膜蛋白折叠的理解才刚刚开始出现。在这一领域的工作将提供一个特别肥沃的途径，为未来的调查使用二维红外方法同位素编辑的跨膜螺旋，暴露平衡动力学和结构安排的耦合残基在其跨膜的空间安排。螺旋间氢键在螺旋-螺旋相互作用的稳定中也很重要，现在已知2D IR对氢键间的相互作用敏感 该核心项目目前的目标是： - 完成了50 fs双光学参量振荡器，以访问从O-H和N-H拉伸区域到酰胺-III的频率。每个脉冲中的大带宽将确保可以在最宽的可能频率范围内记录交叉峰光谱，并且可以清楚地识别两个模式的联合相关性。 - 开发双频技术，用于通过2D IR记录囊泡、胶束和双胞中可溶性和跨膜肽中酰胺-I、C-D、N-H、O-H和酰胺模式之间的邻近性和耦合。 - 双同位素编辑肽和肽聚集体的多同位素异构体的2D IR光谱的理论和处理。 - 介绍高光密度协议双频二维红外光谱允许研究弱C？膜结合螺旋二聚体中H模式与强酰胺模式的耦合。