Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators
Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators
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DOI：
10.1074/jbc.m312846200
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发表时间：
2004-03-26
影响因子：
4.8
通讯作者：
Remington, SJ
中科院分区：
文献类型：
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作者：
Hanson, GT;Aggeler, R;Remington, SJ
目前用于确定细胞中环境氧化还原电位的方法是劳动密集型的，并且通常需要破坏组织。这排除了对由代谢过程或环境影响引起的氧化还原平衡变化的单细胞或真实的时间研究。通过在适当位置用半胱氨酸取代维多利亚水母（Aequorea victoria）绿色荧光蛋白（GFP）上的表面暴露残基以形成二硫键，已经产生了还原-氧化-敏感GFP（roGFP）. roGFP在约400和490 nm处具有两个荧光激发最大值，并且在体外和体内响应于环境氧化还原电位的变化而显示出快速且可逆的荧光比率变化。分别在2.0埃和1.9埃分辨率下对roGFP 2的还原和氧化晶体进行的晶体结构分析揭示了氧化状态下的高度应变的二硫化物和局部化的主链结构变化，这可能是状态依赖性光谱变化的原因。roGFP 1已经靶向HeLa细胞中的线粒体。使用荧光显微镜对这些细胞或使用荧光计在细胞悬浮液中进行荧光测量揭示roGFP 1探针与线粒体氧化还原状态处于动态平衡，并响应于膜可渗透的还原剂和氧化剂。roGFP 1探针报告说，HeLa细胞线粒体中的基质空间高度减少，中点电位接近-360 mV（假设37 ℃时线粒体pH值接近8.0）。其他工作（C。T.杜利，T。M. Dore，G. Hanson，W. C.杰克逊，S. J. Remington和R. Y. Tsien，提交出版），它表明，HeLa细胞的胞质溶胶也是不寻常的减少，但比线粒体基质少一些。
Current methods for determining ambient redox potential in cells are labor- intensive and generally require destruction of tissue. This precludes single cell or real time studies of changes in redox poise that result from metabolic processes or environmental influences. By substitution of surface- exposed residues on the Aequorea victoria green fluorescent protein ( GFP) with cysteines in appropriate positions to form disulfide bonds, reduction- oxidation- sensitive GFPs ( roGFPs) have been created. roGFPs have two fluorescence excitation maxima at about 400 and 490 nm and display rapid and reversible ratiometric changes in fluorescence in response to changes in ambient redox potential in vitro and in vivo. Crystal structure analyses of reduced and oxidized crystals of roGFP2 at 2.0- and 1.9-Angstrom resolution, respectively, reveal in the oxidized state a highly strained disulfide and localized main chain structural changes that presumably account for the state- dependent spectral changes. roGFP1 has been targeted to the mitochondria in HeLa cells. Fluorometric measurements on these cells using a fluorescence microscope or in cell suspension using a fluorometer reveal that the roGFP1 probe is in dynamic equilibrium with the mitochondrial redox status and responds to membrane- permeable reductants and oxidants. The roGFP1 probe reports that the matrix space in HeLa cell mitochondria is highly reducing, with a midpoint potential near - 360 mV ( assuming mitochondrial pH similar to8.0 at 37degreesC). In other work ( C. T. Dooley, T. M. Dore, G. Hanson, W. C. Jackson, S. J. Remington, and R. Y. Tsien, submitted for publication), it is shown that the cytosol of HeLa cells is also unusually reducing but somewhat less so than the mitochondrial matrix.