6MZ3

mCherry pH sensitive mutant - M66T (mCherryTYG)

Summary for 6MZ3

• Entry DOI: 10.2210/pdb6mz3/pdb
• Descriptor: PAmCherry1 protein (2 entities in total)
• Functional Keywords: fluorescent protein, beta barrel, biosensor, luminescent protein
• Biological source: Discosoma sp. (Sea anemone)
• Total number of polymer chains: 1
• Total formula weight: 30560.25

Authors

Haynes, E.P.,Tantama, M. (deposition date: 2018-11-03, release date: 2019-10-09, Last modification date: 2024-10-30)

Primary citation

Haynes, E.P.,Rajendran, M.,Henning, C.K.,Mishra, A.,Lyon, A.M.,Tantama, M.
Quantifying Acute Fuel and Respiration Dependent pH Homeostasis in Live Cells Using the mCherryTYG Mutant as a Fluorescence Lifetime Sensor.
Anal.Chem., 91:8466-8475, 2019

PubMed Abstract: Intracellular pH plays a key role in physiology, and its measurement in living specimens remains a crucial task in biology. Fluorescent protein-based pH sensors have gained widespread use, but there is limited spectral diversity for multicolor detection, and it remains a challenge to measure absolute pH values. Here we demonstrate that mCherryTYG is an excellent fluorescence lifetime pH sensor that significantly expands the modalities available for pH quantification in live cells. We first report the 1.09 Å X-ray crystal structure of mCherryTYG, exhibiting a fully matured chromophore. We next determine that it has an extraordinarily large dynamic range with a 2 ns lifetime change from pH 5.5 to 9.0. Critically, we find that the sensor maintains a p K of 6.8 independent of environment, whether as the purified protein in solution or expressed in live cells. Furthermore, the lifetime measurements are robustly independent of total fluorescence intensity and scatter. We demonstrate that mCherryTYG is a highly effective sensor using time-resolved fluorescence spectroscopy on live-cell suspensions, which has been previously overlooked as an easily accessible approach for quantifying intracellular pH. As a red fluorescent sensor, we also demonstrate that mCherryTYG is spectrally compatible with the ATeam sensor and EGFP for simultaneous dual-color measurements of intracellular pH, ATP, and extracellular pH. In a proof-of-concept, we quantify acute respiration-dependent pH homeostasis that exhibits a stoichiometric relationship with the ATP-generating capacity of the carbon fuel choice in E. coli. Broadly speaking, our work presents a previously unemployed methodology that will greatly facilitate continuous pH quantification.

PubMed: 31247720
DOI: 10.1021/acs.analchem.9b01562

Experimental method

X-RAY DIFFRACTION (1.088 Å)