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Rational Engineering of an Improved Genetically Encoded pH Sensor Based on Superecliptic pHluorin
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- Yi Shen
- Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada
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- Yurong Wen
- Department of Biochemistry, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada
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- Silvia Sposini
- CNRS, Interdisciplinary Institute for Neuroscience, UMR 5297, University of Bordeaux, Bordeaux 33076, France
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- Anjali Amrapali Vishwanath
- Institut du Cerveau-Paris Brain Institute-ICM, Inserm, CNRS, APHP, Häpital de la Pitié Salpêtrière, Sorbonne Université, 75013 Paris, France
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- Ahmed S. Abdelfattah
- Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virgina 20147, United States
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- Eric R. Schreiter
- Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virgina 20147, United States
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- M. Joanne Lemieux
- Department of Biochemistry, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada
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- Jaime de Juan-Sanz
- Institut du Cerveau-Paris Brain Institute-ICM, Inserm, CNRS, APHP, Häpital de la Pitié Salpêtrière, Sorbonne Université, 75013 Paris, France
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- David Perrais
- CNRS, Interdisciplinary Institute for Neuroscience, UMR 5297, University of Bordeaux, Bordeaux 33076, France
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- Robert E. Campbell
- Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada
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Description
Genetically encoded pH sensors based on fluorescent proteins are valuable tools for the imaging of cellular events that are associated with pH changes, such as exocytosis and endocytosis. Superecliptic pHluorin (SEP) is a pH-sensitive green fluorescent protein (GFP) variant widely used for such applications. Here, we report the rational design, development, structure, and applications of Lime, an improved SEP variant with higher fluorescence brightness and greater pH sensitivity. The X-ray crystal structure of Lime supports the mechanistic rationale that guided the introduction of beneficial mutations. Lime provides substantial improvements relative to SEP for imaging of endocytosis and exocytosis. Furthermore, Lime and its variants are advantageous for a broader range of applications including the detection of synaptic release and neuronal voltage changes.
Journal
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- ACS Sensors
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ACS Sensors 8 (8), 3014-3022, 2023-07-23
American Chemical Society (ACS)
