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利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 用双频二维红外光谱方法研究了不同环境中多肽模式的二维红外光谱。在这种方法中，两个感兴趣的模式都被合并到同一个非线性信号中，因此只有当它们以某种方式耦合时，它们的联合信号才存在。我们介绍的另一种方法是双同位素置换，这是一种通过2D IR揭示结构接近的策略。使用这些方法取得的成功的初步结果促使人们进行了更雄心勃勃的实验，可以回答新类型的问题。需要双频方法，因为红外激光脉冲的带宽太窄，无法同时访问广泛分离的振动模式。在双频实验方面，已报告了足够多的例子，表明该方法具有巨大的潜力，但该方法尚处于起步阶段。在2D IR实验中，只有几个频率被纳入实验，并且主要是获得了强肽主链模式。首次用泵浦/探头2D IR方法的双频结果很好地显示了二肽和N-甲基乙酰胺的N-H和C=O基团之间的偶联。还报道了由两个频率相互作用的多肽产生的外差信号的结果。该方法被应用于二肽，最近还被应用于模拟系统，当弱跃迁与强跃迁耦合时，该系统戏剧性地放大了信号。这些实验提供了探索传统方法不容易获得的多肽结构和动力学细节的机会。不仅可以访问覆盖广泛频率范围的单独的酰胺模式，而且工程设计的探测器，例如那些在水的透明区域中含有CN基团的探测器，可以提供一组新的结构约束。在另一个例子中，碳氢键的选择性氢化可以暴露C-D键，用于2D IR双频分析，如下一段所讨论的。 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、转运体和氧化还原蛋白等。许多都有一个单一的跨膜(TM)螺旋，它与其他TM螺旋结合形成螺旋束。这些组装体发生在各种生物情况下，对于研究膜中的折叠也具有优势。尽管人们对它们产生了浓厚的兴趣，但它们的3D结构和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晶体存在固有的困难，而且它们在溶液核磁共振研究中的溶解性很差。血糖素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。该结构域负责蛋白质的二聚化，只有少数残基构成二聚化界面。 2D IR方法也将被配置为获得稳定膜蛋白折叠构象的特征。在螺旋膜蛋白的折叠过程中，驱动力可能是分散力、相互作用和/或膜内形成的强氢键。对于水溶性蛋白质，将埋藏的非极性侧链改变为更小的侧链是有能量成本的。对胶束中膜蛋白折叠的了解才刚刚开始。这一领域的工作将为未来使用2D IR方法对同位素编辑的跨膜螺旋进行研究提供特别有利的途径，这些螺旋揭示了耦合残基在膜上的空间排列方面的平衡动力学和结构排列。螺旋间氢键在螺旋-螺旋相互作用的稳定中也很重要，目前已知2D IR对跨氢键的相互作用很敏感 该核心项目的当前目标是： -完成50飞秒双光参量振荡器，以访问从O-H和N-H向下延伸到约10的酰胺-III区域的频率，以实现双频2D IR。每个脉冲的大带宽将确保可以在尽可能宽的频率范围内记录交叉峰谱，并且可以清楚地识别两个模式的联合关联。 -开发双频技术，通过2D IR记录囊泡、胶束和双胶束中可溶和跨膜多肽中的酰胺-I、C-D、N-H、O-H和酰胺模式之间的接近和偶联。 -双同位素编辑多肽和多肽聚集体的多个同位素聚集体的二维红外光谱的理论和处理。 -将高密度协议引入双频2D IR光谱，允许研究膜结合的螺旋二聚体中弱C？H模与强酰胺模的耦合。