IN VIVO FLUORESCENCE FLUCTUATION SPECTROSCOPY

体内荧光涨落光谱

• 批准号: 7175456
• 负责人: JOACHIM D MUELLER
• 金额: $ 20.21万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-02-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7175456
• 关键词: Accounting; Affect; Algorithms; Area; Basic Science; Binding; Biological; Cell physiology; Cells; Cellular biology; Chimeric Proteins; Color; Complex; Condition; Count; Data; Detection; Development; Diabetes Mellitus; Diffusion; Disease; Dissociation; Environment; Equilibrium; Event; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescence Spectroscopy; Gene Expression; Genetic Transcription; Goals; Green Fluorescent Proteins; In Vitro; Investigation; Kinetics; Label; Life; Ligands; Malignant Neoplasms; Measurement; Measures; Metabolism; Methods; Molecular; NCOA2 gene; Nuclear Receptors; Performance; Pharmacologic Substance; Photons; Physiological; Preclinical Drug Evaluation; Process; Property; Proteins; Protocols documentation; RXR; Rage; Range; Recruitment Activity; Reproduction; Research; Research Personnel; Resolution; Spectrum Analysis; Stimulus; System; Techniques; Testing; Time; Tube; Variant; detector; fighting; fluorophore; improved; in vivo; interest; new technology; programs; protein protein interaction; research study; response; single molecule; statistics; submicron; theories; tool; transcription factor; two-photon

DESCRIPTION (provided by applicant): The unique features of fluorescence fluctuation spectroscopy (FFS) make this technique attractive for cellular applications. FFS determines kinetic and molecular properties of proteins with submicron resolution and single molecule sensitivity. Especially, the application of FFS to proteins tagged with a variant of green fluorescent protein has the potential to revolutionize in vivo studies. We will focus here on dual-color photon counting histogram (PCH) analysis. PCH is a relatively new FFS technique with the potential to provide quantitative information about protein association in living cells. Dual-color PCH exploits two fluorescent protein labels with distinctively different emission spectra. Separating the two colors into different detection channels strongly increases the sensitivity of PCH in detecting protein association. We will adept dual-color PCH for in vivo studies, implement global analysis methods and thoroughly characterize the technique. The long-term objective of the proposed research lies in the concurrent development and application of fluorescence fluctuation techniques, so that their full potential for in vivo studies is realized. The impact of this new technology will be felt in many biological areas with applications ranging from basic research in cell biology to pharmaceutical drug screening. Dual-color PCH will be applied to study the interactions between nuclear receptors and their coregulators in vivo. Nuclear receptors are transcription factors that turn gene expression on and off. They inhibit or enhance transcription by recruiting an array of coactivat6r or corepressor proteins. The quantitative characterization of the oligomerization state of the receptorcoregulator complex by dual-color PCH will be at the focus of this study. Nuclear receptors affect processes as diverse as reproduction, development, and general metabolism, and have been implicated in cancer, diabetes, and many other diseases. Dual-color PCH could help in fighting these diseases by providing detailed information about the protein interactions that govern gene expression in cells.

描述（由申请人提供）： 荧光涨落光谱（FFS）的独特功能，使这项技术有吸引力的细胞应用。FFS以亚微米分辨率和单分子灵敏度测定蛋白质的动力学和分子特性。特别是，FFS的应用程序与绿色荧光蛋白的变体标记的蛋白质有可能彻底改变在体内的研究。我们将重点介绍双色光子计数直方图（PCH）分析。PCH是一种相对较新的FFS技术，具有提供活细胞中蛋白质缔合的定量信息的潜力。双色PCH利用具有明显不同的发射光谱的两种荧光蛋白标记。将这两种颜色分离到不同的检测通道中大大增加了PCH检测蛋白质缔合的灵敏度。我们将熟练的双色PCH在体内研究，实施全球分析方法和彻底的技术特点。拟议研究的长期目标在于同时开发和应用荧光波动技术，使其在体内研究的全部潜力得以实现。这项新技术的影响将在许多生物领域感受到，其应用范围从细胞生物学的基础研究到药物筛选。双色PCH将用于研究核受体与其辅调节因子之间的相互作用。核受体是开启和关闭基因表达的转录因子。它们通过募集一系列共激活蛋白或共阻遏蛋白来抑制或增强转录。定量表征的receptorcoregulator复合物的寡聚化状态的双色PCH将在本研究的重点。核受体影响生殖、发育和一般代谢等多种过程，并与癌症、糖尿病和许多其他疾病有关。双色PCH可以通过提供有关控制细胞中基因表达的蛋白质相互作用的详细信息来帮助对抗这些疾病。