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.

Environmental Effects on Reactive Oxygen Species Detection—Learning from the Phagosome

“…In the case of OxyFRET and PerFRET, the detection relies on the use of Cerulean (pK a = 5.2) (23) and cp173Venus (pK a of Venus is 6, the one of cp173Venus should be slightly higher) (64). We recently evaluated the influence of pH on donor fluorescence lifetime and ratiometric signals of FRET biosensors (6). The pH sensitivities of the donor and the acceptor contribute both to the variations of FRET efficiency with pH.…”

“…At pHs lower than about two pH units below pK a , the fluorescence itself and its variations can be significantly decreased (4,9,21,22). Green and yellow FPs (GFP, YFP, Venus, Citrine) that bear a tyrosine in their chromophore have pK a values between pH 5.7 and 6.9 (64) that may increase in presence of high chloride concentrations (6). The pK a is also increased for the circularly permutated version of a FP, whose barrel has been modified in front of the chromophore.…”

“…In addition, if both intensities ( and ) drop in the same proportion, their ratio which is the readout for ratiometric FRET sensing might be kept constant. The absence of signal variations hides a considerable drop of the FRET-based sensing as the FRET itself almost disappeared (6). As a consequence, at low pH, the biosensor is unable to report any biochemical changes in its environment.…”

Fluorescent Reporters and Biosensors for Probing the Dynamic Behavior of Protein Kinases

“…Since the initial cloning of GFP, a wide variety of autofluorescent protein derivatives have been developed and optimized to improve brightness, quantum yield and pH-insensitivity, so as to yield donor/acceptor couples that may transfer fluorescence resonance energy as efficiently as possible within single-chain FRET biosensors [28,79,80,81,82,83,84,85,86,87]. Although several different AFP pairs have been used for FRET biosensors, the most widely used FRET couples employed for PK biosensors are undoubtedly YFP/CFP and EGFP/mRFP or Cherry.…”

“…Ectopic expression requires time and may lead to significant differences in expression levels in a cellular population, unless cells are stably transfected. Moreover, many AFPs exhibit/suffer from strong pH sensitivities close to the physiological pH range, which may have dramatic consequences on fluorescence signals measured in living cells [87]. They may further be subject to oxidation, misfolding, aggregation or oligomerization, thereby failing to fluorescence, leading to decreased, unreliable or misleading interpretations [131].…”