Genetic engineering of circularly permuted yellow fluorescent protein reveals intracellular acidification in response to nitric oxide stimuli

Haitao Deng, Jingyi Li, Yao Zhou, Yang Xia, Chao Chen, Zhemin Zhou, Hui Wu, Ping Wang, Shengmin Zhou

Research output: Contribution to journalArticlepeer-review

5 Scopus citations


Intracellular pH (pHi) is a crucial parameter in cell biology; thus, a series of pH probes have been developed to determine pHi changes in living cells. However, more sensitive and non-perturbing ratiometric pH probes are needed for accurate pHi measurements. While the fluorescence of circular permutated YFP (cpYFP) is hypersensitive to pH changes due to its intrinsic properties, the single excitation peak of this protein restricts its capacity of becoming a rational type of pH sensor. Herein, we collected several cpYFP-based probes with dual excitation peaks and constructed their corresponding loss-of-function mutants to screen for a potential competent pH probe. The most sensitive probe was named NocPer. NocPer consists of cpYFP inserted into inactive-mutated GAF and AAA+, which are two regulatory domains of E. coli NorR, a nitric oxide (NO)-specific transcription factor. Fluorescence emission of NocPer peaks at 517 nm while exhibiting dual excitation peaks at 420 and 495 nm, which can be used for ratiometric imaging. This new pH sensor has a large ratio response dynamic (pH range of 7.0–11.0), which covers the physiological pH range (pH 7.0–8.0), and exhibits an approximately 3-fold higher fluorescent signal in response to a pH increase from 7.0 to 8.0 than that of pHluorin. Using NocPer, we discovered a new biological phenomenon in which NO exposure decreases the E. coli pHi, which led to the hypothesis that pathogens decrease their own pHi during infection. Further, we elucidated that the NO-induced inhibition of cytochrome c oxidase in the respiratory chain is responsible for the decline in pHi, which might represent a protective strategy of E. coli under NO stress conditions. Our results demonstrated that NocPer is a ratiometric pH probe with high sensitivity for the physiological pH range.

Original languageEnglish (US)
Article number101943
JournalRedox Biology
StatePublished - May 2021

Bibliographical note

Funding Information:
This study was funded by the National Major Science and Technology Projects of China ( 2019ZX09739001 ), the National Natural Science Foundation of China ( 22077032 , 21672065 and 21636003 ), the Project Funded by the International S&T Innovation Cooperation Key Project ( 2017YFE0129600 ), the Fundamental Research Funds for the Central Universities ( 22221818014 ), and the 111 Project ( B18022 ).

Publisher Copyright:
© 2021 The Authors


  • Fluorescent probe
  • Nitric oxide
  • cpYFP
  • pH indicator


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