Rational Engineering of an Improved Genetically Encoded pH Sensor Based on Superecliptic pHluorin

Yi Shen, Yurong Wen, Silvia Sposini, Anjali Amrapali Vishwanath, Ahmed S. Abdelfattah, Eric R. Schreiter, M. Joanne Lemieux, Jaime de Juan-Sanz, David Perrais, Robert E. Campbell
ACS Sens.. 2023-07-23; 8(8): 3014-3022
DOI: 10.1021/acssensors.3c00484

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Shen Y(1), Wen Y(2)(3), Sposini S(4)(5), Vishwanath AA(6), Abdelfattah AS(7)(8), Schreiter ER(7), Lemieux MJ(2), de Juan-Sanz J(6), Perrais D(4), Campbell RE(1)(9).

Author information:
(1)Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada.
(2)Department of Biochemistry, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
(3)Center for Microbiome Research of Med-X Institute, The First Affiliated Hospital, Xi’an Jiaotong University, Xi’an, Shaanxi 710061, China.
(4)CNRS, Interdisciplinary Institute for Neuroscience, UMR 5297, University of Bordeaux, Bordeaux 33076, France.
(5)Department of Metabolism, Digestion and Reproduction, Institute of Reproductive and Developmental Biology, Imperial College London, London SW7 2BX, United Kingdom.
(6)Institut du Cerveau-Paris Brain Institute-ICM, Inserm, CNRS, APHP, Häpital de la Pitié Salpêtrière, Sorbonne Université, 75013 Paris, France.
(7)Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virgina 20147, United States.
(8)Department of Neuroscience, Brown University, Providence, Rhode Island 02906, United States.
(9)Department of Chemistry, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan.

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.


Auteurs Bordeaux Neurocampus