title: pH sensitivity of FRET reporters based on cyan and yellow fluorescent proteins.
abstract: It is generally acknowledged that the popular cyan and yellow fluorescent proteins carried by genetically encoded reporters suffer from strong pH sensitivities close to the physiological pH range. We studied the consequences of these pH responses on the intracellular signals of model Förster resonant energy transfer (FRET) tandems and FRET-based reporters of cAMP-dependent protein kinase activity (AKAR) expressed in the cytosol of living BHK cells, while changing the intracellular pH by means of the nigericin ionophore. Although the simultaneous pH sensitivities of the donor and the acceptor may mask each other in some cases, the magnitude of the perturbations can be very significant, as compared to the functional response of the AKAR biosensor. Replacing the CFP donor by the spectrally identical, but pH-insensitive Aquamarine variant (pK1/2 = 3.3) drastically modifies the biosensor pH response and gives access to the acid transition of the yellow acceptor. We developed a simple model of pH-dependent FRET and used it to describe the expected pH-induced changes in fluorescence lifetime and ratiometric signals. This model qualitatively accounts for most of the observations, but reveals a complex behavior of the cytosolic AKAR biosensor at acid pHs, associated to additional FRET contributions. This study underlines the major and complex impact of pH changes on the signal of FRET reporters in the living cell.
authors: CNRS, Laboratoire de Chimie Physique, UMR 8000 and Université Paris-Sud, Avenue Jean Perrin 15, 91405, Orsay, France.
authors: Dahdjim-Benoît
authors: DB
authors: Betolngar
authors: Marie
authors: M
authors: Erard
authors: Hélène
authors: H
authors: Pasquier
authors: Yasmina
authors: Y
authors: Bousmah
authors: Awa
authors: A
authors: Diop-Sy
authors: Elvire
authors: E
authors: Guiot
authors: Pierre
authors: P
authors: Vincent
authors: Fabienne
authors: F
authors: Mérola
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry*; D002460:Cell Line; D006224:Cricetinae; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007476:Ionophores / Q000494:pharmacology; D008164:Luminescent Proteins / Q000737:chemistry*; D009550:Nigericin / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D007476:Ionophores; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D009550:Nigericin