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M153R mutation in a pH-sensitive green fluorescent protein stabilizes its fusion proteins
Authors:Morimoto Yusuke V  Kojima Seiji  Namba Keiichi  Minamino Tohru
Affiliation:1Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan;2Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-Ku, Nagoya, Japan;3Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama, Japan;J. Craig Venter Institute, United States of America
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.

Methodology/Principal Findings

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/Significance

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.
Keywords:
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