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Proc Natl Acad Sci U S A ; 121(21): e2314604121, 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38748581

RESUMO

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 superfolder green fluorescent protein (GFP) inserted between the ATP-binding helices of the ε-subunit of a bacterial F0-F1 ATPase. Optimizing the linkers joining the two domains resulted in a ~fivefold to sixfold 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 suitable spectrally separated fluorescent protein provides an optional ratiometric readout allowing comparisons of ATP across cellular regions. Subcellular targeting 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.


Assuntos
Trifosfato de Adenosina , Proteínas de Fluorescência Verde , Animais , Humanos , Trifosfato de Adenosina/análise , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Técnicas Biossensoriais/métodos , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/genética , Fosforilação Oxidativa , ATPases Translocadoras de Prótons/metabolismo , ATPases Translocadoras de Prótons/genética
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