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M153R mutation in a pH-sensitive green fluorescent protein stabilizes its fusion proteins

Yusuke V Morimoto1, Seiji Kojima, Keiichi Namba

  • 1Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan.

Plos One
|May 12, 2011
PubMed
Abstract

Insights

A new mutation in pHluorin (M153R) stabilizes fusion proteins, improving fluorescent imaging. This pHluorin(M153R) tool enhances biological process analysis and pH measurement in living cells.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • Green fluorescent protein (GFP) is widely used in biological studies.
  • Proteolytic cleavage of GFP fusion proteins compromises signal quality and data interpretation.

Purpose of the Study:

  • To develop a stabilized pH-sensitive GFP variant for improved biological analysis.
  • To overcome limitations of proteolytic degradation in GFP-based assays.

Main Methods:

  • Introduction of the M153R mutation into the pHluorin protein.
  • Assessment of fusion protein stability and pH sensitivity in vivo.
  • Analysis of fluorescence intensity and excitation ratios at different pH values.

Main Results:

  • The M153R mutation significantly stabilizes pHluorin fusion products.
  • The mutant pHluorin retains pH sensitivity, indicated by the 410/470 nm excitation ratio.
  • M153R mutation enhances in vivo brightness without altering pH-dependent fluorescence ratios.

Conclusions:

  • pHluorin(M153R) serves as a robust tool for direct fusion to target proteins.
  • This probe enables accurate measurement of local pH in living cells.
  • It facilitates improved analysis of subcellular localization of target proteins.

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