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: 8362564
• 负责人: ROBIN Main HOCHSTRASSER
• 金额: $ 13.01万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8362564
• 关键词: Address; Amides; Area; Binding; Biological; Biological Models; Carbon; Cell Surface Receptors; Cell physiology; Coupled; Coupling; Development; Dimerization; Dipeptides; Engineering; Ensure; Environment; Equilibrium; Frequencies; Funding; Future; Glycophorin A; Goals; Grant; Hydrogen Bonding; Individual; Investigation; Ion Channel; Isotopes; Joints; Laboratories; Lasers; Membrane; Membrane Proteins; Methods; Micelles; Molecular Conformation; N-methylacetamide; National Center for Research Resources; Optics; Oxidation-Reduction; Peptides; Physiologic pulse; Principal Investigator; Process; Proteins; Protocols documentation; Pump; Reporting; Research; Research Infrastructure; 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. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供 该子项目的主要支持。 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源的子项目可能列出的总成本。 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 现在已经通过双频 2D IR 检查了各种环境中肽模式的 2D IR 光谱。在该方法中，两种感兴趣的模式都被纳入相同的非线性信号中，因此它们的联合信号仅在它们耦合时才存在。我们引入的另一种方法是双同位素替换，这是一种通过 2D IR 暴露结构邻近性的策略，使用这些方法取得的成功的初步结果促进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，已经报道了足够数量的例子，表明该方法具有巨大的潜力，但该方法具有巨大的潜力。该方法还处于起步阶段。只有少数频率被纳入 2D IR 实验，并且主要是获得了强肽骨架模式，泵/探针 2D IR 方法的第一个双频结果完美地显示了 N-H 之间的耦合。还报道了二肽和 N-甲基乙酰胺的 C=O 基团与两个频率相互作用产生的外差信号。该方法应用于二肽，最近应用于模型系统，该系统在弱时使预期信号放大。这些实验提供了探索传统方法无法轻松获取的肽结构和动力学细节的机会，不仅可以获取而且还可以设计涵盖广泛频率的单个酰胺模式。在另一个例子中，碳氢键的选择性氘化可以暴露用于二维红外双频分析的 C-D 键，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。 开发 2D IR 方法对于研究膜蛋白非常重要，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，许多蛋白具有单跨膜。 TM）螺旋与其他TM螺旋结合形成螺旋束，这些组装体出现在多种生物学情况中，并且对于膜折叠的研究也具有优势，尽管人们对它们很感兴趣。由于生长适合 X 射线衍射的 3D 晶体固有的困难以及它们在溶液 NMR 研究中的溶解度差，对其 3D 结构和动力学的研究仍然具有挑战性。 血型糖蛋白 A (GpA) 螺旋二聚体的 TM 结构域为二维红外图解螺旋关联的结构基础，表明该结构域负责蛋白质二聚化，并且只有少数残基构成二聚化界面。 2D IR 方法还将被配置为获取稳定膜蛋白折叠构象的特征，在螺旋膜蛋白的折叠中，驱动力可能是色散力相互作用和/或与水溶性膜中形成的强氢键。将埋藏的非极性侧链改变为较小的侧链需要消耗能量，对胶束中膜蛋白折叠的理解才刚刚开始，这将为未来的研究提供一条特别丰富的途径。对同位素编辑的跨膜螺旋进行 2D IR 方法，可以揭示耦合残基在跨膜空间排列方面的平衡动力学和结构排列，对于稳定螺旋-螺旋相互作用也很重要，并且 2D IR 也很重要。现在已知对氢键相互作用敏感 该核心项目当前的目标是： - 完成 50 fs 双光学参量振荡器，以获取从 O-H 和 N-H 延伸区域到约 10 的酰胺-III 的频率，以实现双频 2D IR。每个脉冲的大带宽将确保交叉峰值光谱。可以在尽可能宽的频率范围内进行记录，并且可以清楚地识别两种模式的联合相关性。 - 开发双频技术，通过 2D IR 记录囊泡、胶束和双束中可溶性和跨膜肽中的酰胺-I、C-D、N-H、O-H 和酰胺模式之间的接近度和耦合。 - 双同位素编辑肽和肽聚集体的多种同位素异构体的 2D IR 光谱的理论和处理。 - 将高光密度协议引入双频 2D IR 光谱，可以研究弱 C¿ H 模式与膜结合螺旋二聚体中的强酰胺模式偶联。