title: M153R mutation in a pH-sensitive green fluorescent protein stabilizes its fusion proteins.
abstract: BACKGROUND
  Green fluorescent protein (GFP) and its fusion proteins have been used extensively to monitor and analyze a wide range of biological processes. However, proteolytic cleavage often removes GFP from its fusion proteins, not only causing a poor signal-to-noise ratio of the fluorescent images but also leading to wrong interpretations.
  
  
  RESULTS
  Here, we report that the M153R mutation in a ratiometric pH-sensitive GFP, pHluorin, significantly stabilizes its fusion products while the mutant protein still retaining a marked pH dependence of 410/470 nm excitation ratio of fluorescence intensity. The M153R mutation increases the brightness in vivo but does not affect the 410/470-nm excitation ratios at various pH values.
  
  
  CONCLUSIONS
  Since the pHluorin(M153R) probe can be directly fused to the target proteins, we suggest that it will be a potentially powerful tool for the measurement of local pH in living cells as well as for the analysis of subcellular localization of target proteins.
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan.
authors.forename: Yusuke V
authors.initials: YV
authors.lastname: Morimoto
authors.forename: Seiji
authors.initials: S
authors.lastname: Kojima
authors.forename: Keiichi
authors.initials: K
authors.lastname: Namba
authors.forename: Tohru
authors.initials: T
authors.lastname: Minamino
mesh_terms: D001483:Base Sequence; D005821:Genetic Techniques; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D008856:Microscopy, Fluorescence / Q000379:methods; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D019779:Salmonella enterica / Q000235:genetics*; D017422:Sequence Analysis, DNA; D013696:Temperature
chemical_list: C485187:PHluorin; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins