iATPSnFR2: A high-dynamic-range fluorescent sensor for monitoring intracellular ATP.
Proc Natl Acad Sci U S A
; 121(21): e2314604121, 2024 May 21.
Article
in En
| MEDLINE
| ID: mdl-38748581
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 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.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Adenosine Triphosphate
/
Green Fluorescent Proteins
Limits:
Animals
/
Humans
Language:
En
Journal:
Proc Natl Acad Sci U S A
Year:
2024
Document type:
Article