New Red-Emitting Chloride-Sensitive Fluorescent Protein with Biological Uses Salto González, Rafael Girón González, María Dolores Puente Muñoz, Virginia Vílchez Rienda, José Dámaso Espinar Barranco, Laura Valverde Pozo, Javier Paredes Martínez, José Manuel Red fluorescent proteins Chloride sensors Imaging Two-photon excitation microscopy Genetically encoded sensors Intracellular sensors This work was financially supported by CTQ2017-85685-R, CTQ2017-85454-C2-1-P, and CTQ2017-86125-P (MICIU/AEI/ERDF). J.M.P. and J.D.V. thank UEQ for funding. J.V.P. is supported by an FPU fellowship (FPU17/04749). D.A. was supported by the Italian Cystic Fibrosis Foundation (grant FFC#3/2019) and by Fondazione Cassa Rurale Trento Rovereto (ref 2018.256). The authors acknowledge the Universidad of Granada (Spain) cell culture and microscopy central facilities (CIC-UGR). A new chloride-sensitive red fluorescent protein derived from Entacmaea quadricolor is described. We found that mBeRFP exhibited moderate sensitivity to chloride and, via sitedirected mutagenesis (S94V and R205Y), we increased the chloride affinity by more than an order of magnitude (kd = 106 ± 6 mM) at physiological pH. In addition, cis−trans isomerization of the chromophore produces a dual emission band with different chloride sensitivities, which allowed us to develop a ratiometric methodology to measure intracellular chloride concentrations. 2022-02-11T07:55:18Z 2022-02-11T07:55:18Z 2021-06-21 journal article ACS Sens. 2021, 6, 2563−2573. [https://doi.org/10.1021/acssensors.1c00094] http://hdl.handle.net/10481/72792 10.1021/acssensors.1c00094 eng http://creativecommons.org/licenses/by/3.0/es/ open access Atribución 3.0 España American Chemical Society