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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 与典型批量实验中的整体平均值相比，单分子光谱在研究分子特性分布方面具有巨大优势。我们开发了各种装置，可以与实验室中可用的所有激励源相结合，并提供频率和时间分辨检测以及成像功能。我们的主要研究方向是生理条件下蛋白质动力学和蛋白质折叠的研究以及单细胞内分子过程的研究。 我们将继续研究各种固定化技术，进一步研究凝胶、溶胶-凝胶、海藻糖玻璃、反胶束、γ-环糊精、沸石笼和胶束在水环境中固定单一蛋白质和酶。 我们正在获取用于测量这些环境中的结构波动、蛋白质折叠/展开和模型肽（螺旋线圈转变）、酶反应和钙离子信号传导（钙调蛋白）的协议。通过监测构象变化以及温度、pH、盐和离子扩散的影响的实验，还可以了解水在封装中的作用、空腔中水含量的变化、蛋白质水的封装以及蛋白质与周围环境的相互作用。空腔正在研究中。 我们最近研究的目的是对具有一定曲率范围的各种脂质、胆固醇、去污剂和头基的膜中螺旋聚集的缔合和解离、扩散和动态效应的异质性进行定量评估。这些实验通过单分子荧光寿命和针对特定标记和环境敏感探针的 FRET 实验，实现血型糖蛋白 A (GpA) 和其他 TM 蛋白跨膜 (TM) 部分的结构波动和分布的可视化。 另一个目标是通过环境敏感探针的单分子荧光相关实验来定量描述钙调蛋白的钙信号传导机制的异质性。 针对膜、囊泡和脂质双层中脱辅基肌红蛋白和细胞色素-c 的构象状态分布和自由能表面的实验正在进行中。探测荧光寿命分布、FRET 和环境敏感响应的单分子方法，为解决构象运动的异质性、蛋白质折叠和展开特性以及自由能表面的问题提供了强大的途径，而这些是从批量实验中无法获得的。