三磷酸腺苷
ATP酶
ATP水解
亚细胞定位
线粒体基质
质子泵
生物化学
生物
胞浆
细胞生物学
生物物理学
化学
酶
基因
作者
Jonathan S Marvin,Alexandros C. Kokotos,Mukesh Kumar,Camila Pulido,Ariana N. Tkachuk,Jian Yao,Timothy A. Brown,Timothy A. Ryan
标识
DOI:10.1101/2023.08.24.554624
摘要
Abstract We developed a significantly improved genetically encoded quantitative adenosine triphosphate (ATP) sensor to provide real-time dynamics of ATP levels in subcellular compartments. iATPSnFR2 is a variant of iATPSnFR1, a previously developed sensor that has circularly permuted super-folder GFP inserted between the ATP-binding helices of the ε -subunit of a bacterial F 0 -F 1 ATPase. Optimizing the linkers joining the two domains resulted in a ∼ 5-6 fold improvement in the dynamic range compared to the previous generation sensor, with excellent discrimination against other analytes and affinity variants varying from 4 μM to 500 μM. A chimeric version of this sensor fused to either the HaloTag protein or a suitably spectrally separated fluorescent protein, provides a ratiometric readout allowing comparisons of ATP across cellular regions. Subcellular targeting of the sensor to nerve terminals reveals previously uncharacterized single synapse metabolic signatures, while targeting to the mitochondrial matrix allowed direct quantitative probing of oxidative phosphorylation dynamics. Significance Statement Adenosine triphosphate (ATP) is a key metabolite necessary for cellular life. Here we develop a next-generation genetically encoded ratiometric fluorescent ATP sensor that allows subcellular tracking of ATP levels in living cells. The large dynamic range makes it possible to follow the dynamics of this metabolite across cells and subcellular regions under different metabolic stressors. We expect that iATPSnFR2 will provide researchers with exciting new opportunities to study ATP dynamics with temporal and spatial resolution that has, until now, been unavailable.
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