Rational Engineering of an Improved Genetically Encoded pH Sensor Based on Superecliptic pHluorin
- Yi Shen*
- Yurong WenYurong WenDepartment of Biochemistry, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, Alberta T6G 2H7, CanadaCenter for Microbiome Research of Med-X Institute, The First Affiliated Hospital, Xi’an Jiaotong University, Xi’an, Shaanxi 710061, ChinaMore by Yurong Wen
- Silvia SposiniSilvia SposiniCNRS, Interdisciplinary Institute for Neuroscience, UMR 5297, University of Bordeaux, Bordeaux 33076, FranceDepartment of Metabolism, Digestion and Reproduction, Institute of Reproductive and Developmental Biology, Imperial College London, London SW7 2BX, United KingdomMore by Silvia Sposini
- Anjali Amrapali VishwanathAnjali Amrapali VishwanathInstitut du Cerveau-Paris Brain Institute-ICM, Inserm, CNRS, APHP, Häpital de la Pitié Salpêtrière, Sorbonne Université, 75013 Paris, FranceMore by Anjali Amrapali Vishwanath
- Ahmed S. Abdelfattah
- Eric R. SchreiterEric R. SchreiterJanelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virgina 20147, United StatesMore by Eric R. Schreiter
- M. Joanne Lemieux
- Jaime de Juan-SanzJaime de Juan-SanzInstitut du Cerveau-Paris Brain Institute-ICM, Inserm, CNRS, APHP, Häpital de la Pitié Salpêtrière, Sorbonne Université, 75013 Paris, FranceMore by Jaime de Juan-Sanz
- David PerraisDavid PerraisCNRS, Interdisciplinary Institute for Neuroscience, UMR 5297, University of Bordeaux, Bordeaux 33076, FranceMore by David Perrais
- , and
- Robert E. Campbell*
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.
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