SPECTROSCOPY OF SINGLE PROTEINS AND BIOLOGICAL ASSEMBLIES: EQUILIBRIUM DYNAMICS

单一蛋白质和生物组装体的光谱学：平衡动力学

• 批准号: 7598432
• 负责人: ROBIN Main HOCHSTRASSER
• 金额: $ 3.97万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7598432
• 关键词: Address; Biological; Calcium Signaling; Calmodulin; Cells; Cholesterol; Computer Retrieval of Information on Scientific Projects Database; Condition; Detection; Detergents; Diffuse; Diffusion; Dissociation; Environment; Enzymes; Equilibrium; Fluorescence; Fluorescence Resonance Energy Transfer; Free Energy; Frequencies; Funding; Gel; Glass; Glycophorin A; Grant; Head; Helix (Snails); Heterogeneity; Image; Imagery; Immobilization; Institution; Investigation; Ions; Label; Lipid Bilayers; Lipids; Measurement; Membrane; Membrane Proteins; Methods; Micelles; Modeling; Molecular; Molecular Conformation; Monitor; Motion; Peptides; Physiological; Process; Property; Protein Dynamics; Proteins; Protocols documentation; Purpose; Range; Reaction; Research; Research Personnel; Resources; Role; Salts; Source; Spectrum Analysis; Surface; Techniques; Temperature; Time; Trehalose; United States National Institutes of Health; Variant; Vesicle; Water; apomyoglobin; aqueous; cyclomalto-octaose; cytochrome c; ear helix; gamma-Cyclodextrins; protein folding; research study; response; single molecule

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. Single molecule spectroscopy offers the great advantages of studying distributions of molecular properties compared to ensemble averages in a typical bulk experiment. We have developed various set ups which can be combined with all excitation sources available in the lab, and which offer frequency- and time-resolved detection as well as imaging capabilities. Our main research direction is the investigation of protein dynamics and protein folding under physiological conditions and the study of molecular processes within single cells. We will continue to investigate various immobilization techniques, further examining gels, sol-gels, trehalose glasses, reverse micelles, gamma-cyclodextrin, zeolite cages and micelles to immobilize single proteins and enzymes in aqueous environments. We are obtaining protocols for measurements of structural fluctuations, folding/unfolding of proteins and model peptides (helix coil transitions), enzyme reactions and calcium ion signaling (calmodulin) in these environments. Also the role of water in encapsulation, the variation of the water content in cavities, the encapsulation of protein water and the interaction of proteins with the surrounding environment through experiments that monitor conformational changes and the effects of temperature, pH, salts and ions diffusing in the cavities are under study. The purpose of our most recent research is to make quantitative assessments of the heterogeneity of association and dissociation, diffusion and dynamic effects of helix aggregation in membranes with various lipids having a range of curvatures, cholesterol, detergents and head groups. The experiments enable the visualization of structural fluctuations and distributions of trans-membrane (TM) sections of Glycophorin-A (GpA) and other TM proteins by single molecule fluorescence lifetime and FRET experiments for specifically labeled and environmentally sensitive probes. Another aim seeks quantitative descriptions of the heterogeneity of the calcium signaling mechanisms of Calmodulin by single molecule fluorescence correlation experiments with environmentally sensitive probes. Experiments aimed at conformational state distributions and free energy surfaces of apomyoglobin and cytochrome-c in membranes, vesicles, and lipid bilayers are all on-going. Single molecule methods that probe fluorescence lifetime distributions, FRET and environment sensitive responses, provide powerful avenues with which to address the heterogeneity of conformational motions, protein folding and unfolding properties and free energy surfaces that are not obtainable from bulk experiments.

该副本是利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，这不一定是调查员的机构。 与典型的散装实验中的集合平均值相比，单分子光谱学提供了研究分子特性分布的很大优势。我们已经开发了各种设置，这些设置可以与实验室中可用的所有激发源结合使用，并提供频率和时间分辨的检测以及成像功能。我们的主要研究方向是研究蛋白质动力学和蛋白质折叠在生理条件下以及对单个细胞内分子过程的研究。 我们将继续研究各种固定技术，进一步检查凝胶，溶胶 - 凝胶，海藻糖玻璃，反向胶束，γ-环糊精，Zeolite笼子和胶束，以使单一蛋白质和酶固定在水中环境中。 我们正在获取用于测量结构波动，蛋白质折叠/展开的方案，并在这些环境中获得模型肽（螺旋线圈过渡），酶反应和钙离子信号传导（钙调蛋白）。还通过监测构象变化以及温度，pH，盐和离子在腔中扩散的构象变化以及在研究中，水在封装中的作用，腔中水含量的变化以及蛋白质的封装以及蛋白质与周围环境的相互作用。 我们最新研究的目的是对膜聚集的关联和解离，扩散和动态作用的异质性进行定量评估，这些螺旋聚集在具有各种脂质的膜中具有一系列曲率，胆固醇，洗涤剂和头部组。该实验通过单分子荧光寿命和FRET实验，可以看到糖蛋白A（GPA）（GPA）和其他TM蛋白的跨膜（TM）切片的结构波动和分布的可视化实验。 另一个目的是通过单分子荧光相关实验与环境敏感的探针进行的，寻求定量描述钙调蛋白钙信号传导机制的异质性。 针对膜，囊泡和脂质双层中，旨在旨在构象状态分布和自由能表面的实验。探测荧光寿命分布，FRET和环境敏感反应的单分子方法提供了强大的途径，以解决构象运动，蛋白质折叠和展开特性以及自由能表面的异质性，这些途径无法从散装实验中获得。