Molecular mechanism of chaperonin-assisted protein folding studied by single molecule imaging.

单分子成像研究伴侣蛋白辅助蛋白质折叠的分子机制。

基本信息

项目摘要

Chaperonin GroEL mediates substrate protein folding in a repeated cycle of ATP-driven GroEL-GroES association-dissociation processes. We have succeeded in detecting the processes at the level of a single molecule using total internal reflection fluorescence microscopy (TIRFM). Our results indicated that this process consists of two timers approximately 3 sec and 5 sec of the rate-limiting steps. In order to gain understating of this two timer process, acid-denatured GFP was used as a substrate protein for GroEL and its folding was observed. The result from this observation demonstrated that the substrate folding was inhibited in the first 3 sec period, and revealed a cis ADP*-complex, that is a new complex existed in the 5 sec rate-limiting step.Furthermore, the existence of a so called football-shaped intermediate complex, consisting of a GroEL bound both sides with GroES, was examined using a new single molecule fluorescence imaging method. In this imaging, nano-holes with 100 nm diameter were fabricated in a metal film deposited on a slide-glass. This was used to localize evanescent field, reducing the fluorescence background, enabling us to image a single fluorescent molecule in the presence of a high concentration of fluorescence molecules. Using this method, processes of Gro-GroES association-dissociation was revealed at the level of a single molecule in the presence of fluorescently labeled 1 μM of GroES.
伴侣蛋白GroEL在atp驱动的GroEL- groes结合-解离过程的重复循环中介导底物蛋白折叠。我们已经成功地利用全内反射荧光显微镜(TIRFM)检测了单分子水平的过程。我们的结果表明,该过程由两个定时器组成,分别为3秒和5秒的速率限制步骤。为了更好地理解这两个时间过程,酸变性GFP被用作GroEL的底物蛋白,并观察其折叠。结果表明,底物折叠在前3秒被抑制,并在5秒限速步骤中发现一个顺式ADP*复合物,即一个新的复合物。此外,用一种新的单分子荧光成像方法检测了所谓的足球状中间复合物的存在,该复合物由两侧与GroES结合的GroEL组成。在这张图像中,直径为100纳米的纳米孔被制作在沉积在玻片上的金属薄膜中。这被用来定位消失场,减少荧光背景,使我们能够在高浓度荧光分子存在的情况下成像单个荧光分子。利用该方法,在荧光标记的1 μM的GroES存在下,在单分子水平上揭示了Gro-GroES缔合-解离过程。

项目成果

期刊论文数量(102)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Funatsu, T. et al.: "Rapid and sensitive detection method of a bacterium using GFP reporter phage"Microbiol.Immuno.. 46. 365-369 (2002)
Funatsu, T. 等人:“使用 GFP 报告噬菌体的细菌快速灵敏检测方法”Microbiol.Immuno.. 46. 365-369 (2002)
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    0
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High-performance liquid chromatographic assay of N^G-monomethly-L-arginine, N^G, N^G-dimethyl-L-arginine, and N^G, N-<G'>-dimethyl-L-arginine using 4-fluoro-7-nitro-2,1,3-benzoxadiazole as a fluorescent reagent
使用 4 高效液相色谱法测定 N^G-单甲基-L-精氨酸、N^G、N^G-二甲基-L-精氨酸和 N^G、N-<G>-二甲基-L-精氨酸
  • DOI:
  • 发表时间:
    2005
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  • 影响因子:
    0
  • 作者:
    Nonaka;S.;M.Tsunoda;K.Imai;T.Funatsu
  • 通讯作者:
    T.Funatsu
Kinetics and binding sites for interaction of prefoldin with group II chaperonin : contiguous non-native substrate and chaperonin binding sites in archaeal prefoldin
前折叠蛋白与 II 组伴侣蛋白相互作用的动力学和结合位点:古细菌前折叠蛋白中的连续非天然底物和伴侣蛋白结合位点
  • DOI:
  • 发表时间:
    2004
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  • 影响因子:
    0
  • 作者:
    Okochi;M.;T.Nomura;T.Zako;R.Iizuka;H.Ueda;T.Funatsu;M.Leroux;M.Yohda
  • 通讯作者:
    M.Yohda
Preferential immobilization of biomolecules on silicon microstructure array by means of electron beam lithography on organosilane self-assembled monolayer resist
电子束光刻在有机硅烷自组装单层抗蚀剂上将生物分子优先固定在硅微结构阵列上
  • DOI:
  • 发表时间:
    2004
  • 期刊:
  • 影响因子:
    0
  • 作者:
    Tanii;T.;T.Hosaka;T.Miyake;G.Zhang;T.Zako;T.Funatsu;I.Ohdomari
  • 通讯作者:
    I.Ohdomari
Fujiwara, I. et al.: "Microscopic analysis of polymerization dynamics with individual actin filaments"Nature Cell Biol.. 4. 666-673 (2002)
Fujiwara, I. 等人:“用单个肌动蛋白丝进行聚合动力学的微观分析”Nature Cell Biol.. 4. 666-673 (2002)
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FUNATSU Takashi其他文献

FUNATSU Takashi的其他文献

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{{ truncateString('FUNATSU Takashi', 18)}}的其他基金

Manipulating illumination and imaging systems of an optical microscope using a spatial light modulator
使用空间光调制器操纵光学显微镜的照明和成像系统
  • 批准号:
    25650046
  • 财政年份:
    2013
  • 资助金额:
    $ 10.82万
  • 项目类别:
    Grant-in-Aid for Challenging Exploratory Research
Single-molecule analysis of functions of biomolecules by linear zero-mode waveguides
通过线性零模波导对生物分子功能进行单分子分析
  • 批准号:
    24370061
  • 财政年份:
    2012
  • 资助金额:
    $ 10.82万
  • 项目类别:
    Grant-in-Aid for Scientific Research (B)
Single-molecule analysis of functions of biomolecular machines that synthesize or assist folding of proteins.
对合成或协助蛋白质折叠的生物分子机器的功能进行单分子分析。
  • 批准号:
    21370065
  • 财政年份:
    2009
  • 资助金额:
    $ 10.82万
  • 项目类别:
    Grant-in-Aid for Scientific Research (B)
Analyses of functions and interactions of biomolecules by microand nano-devices
利用微米和纳米器件分析生物分子的功能和相互作用
  • 批准号:
    17201031
  • 财政年份:
    2005
  • 资助金额:
    $ 10.82万
  • 项目类别:
    Grant-in-Aid for Scientific Research (A)
Production of novel proteins using the micro system that permits sorting and functional evaluation at single molecular level
使用允许在单分子水平上进行分类和功能评估的微系统生产新型蛋白质
  • 批准号:
    13558088
  • 财政年份:
    2001
  • 资助金额:
    $ 10.82万
  • 项目类别:
    Grant-in-Aid for Scientific Research (B)
Single-molecule imaging of processing and transport of mRNA within a living cell
活细胞内 mRNA 加工和运输的单分子成像
  • 批准号:
    11480196
  • 财政年份:
    1999
  • 资助金额:
    $ 10.82万
  • 项目类别:
    Grant-in-Aid for Scientific Research (B)
Development of Fluorescence Microscopy for Visualizing Single Biomolecules in Living Cells
用于可视化活细胞中单个生物分子的荧光显微镜的发展
  • 批准号:
    10558110
  • 财政年份:
    1998
  • 资助金额:
    $ 10.82万
  • 项目类别:
    Grant-in-Aid for Scientific Research (B)

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Understanding the coordination of DNA mismatch repair using live-cell single-molecule imaging
使用活细胞单分子成像了解 DNA 错配修复的协调
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    BB/Y001567/1
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    2024
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Understanding dual filament regulation in muscle using single molecule imaging in vitro and in myofibrils
使用体外单分子成像和肌原纤维了解肌肉中的双丝调节
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    2024
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Enhancing cellulase activity through single-molecule imaging and protein engineering as a testbed for understanding and improving enzymatic deconstruction of insoluble substrates
通过单分子成像和蛋白质工程增强纤维素酶活性,作为理解和改进不溶性底物酶解构的测试平台
  • 批准号:
    2301377
  • 财政年份:
    2023
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    $ 10.82万
  • 项目类别:
    Standard Grant
Single-Molecule Imaging of Ubiquitination Dynamics in Neurons
神经元泛素化动力学的单分子成像
  • 批准号:
    10817362
  • 财政年份:
    2023
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Acquisition of Two-Color 3-D Minflux for Live-Cell Single Molecule Imaging and Tracking at Unprecedented Spatial and Temporal Resolution
采集双色 3-D Minflux,以前所未有的空间和时间分辨率进行活细胞单分子成像和跟踪
  • 批准号:
    2241074
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    2023
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Mapping Protein Glycosylation by High-Resolution Single Molecule Imaging
通过高分辨率单分子成像绘制蛋白质糖基化图谱
  • 批准号:
    BB/W017024/1
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    2023
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Single Molecule Imaging of RIM1 Scaffold in Photoreceptor Neurotransmission
RIM1 支架在光感受器神经传递中的单分子成像
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    10334878
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    2022
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Ultralong-term single-molecule imaging of amyloid precursor protein (APP) processing in Alzheimer's disease
阿尔茨海默病中淀粉样前体蛋白(APP)加工的超长期单分子成像
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    10738516
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CAREER: Dissecting the Mechanism of Replication Initiation in Vertebrates via Single Molecule Imaging
职业:通过单分子成像剖析脊椎动物复制起始机制
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    2144481
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Investigation of molecular insight into genome editing tools by single-molecule imaging
通过单分子成像研究基因组编辑工具的分子洞察
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    21H01771
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