Fluorescence photoconversion kinetics in novel green fluorescent protein pH sensors (pHluorins)

Genetically encoded pH-sensitive green fluorescent proteins (GFPs) offer significant control over localization, spectral properties, and environmental sensitivity for cell biology and neuroscience applications. Quantitative analysis of biological applications and full exploitation of novel GFP properties rely on a fundamental understanding of their molecular photophysics. In this contribution, the ground- and excited-state fluorescence properties of Ecliptic and Ratiometric pHluorins, two pH-sensitive GFP probes of presynaptic activity, are presented and compared with Sapphire, a neutral phenol GFP mutant. Molecular dynamics have been characterized using fluorescence correlation spectroscopy (FCS) and time-correlated single-photon counting (TCSPC) as a function of pH, excitation wavelength (including 405 nm), detection wavelength, and illumination intensity. As a cellular pH indicator, Ecliptic (EcGFP) is particularly well-suited to the physiological pH range (pK_a = 7.2 ± 0.2) compared with Sapphire (H9; pK_a = 5.7 ± 0.1) and a number of other GFPs. However, both EcGFP and H9 exhibit complex, intensity- and wavelength-dependent partitioning between bright and dark EcGFP states, which may complicate their use in quantitative intracellular measurements. Furthermore, the protonation and deprotonation rate constants for the EcGFP chromophore may limit the ability to resolve pH jumps occurring on time scales faster than ∼0.5 ms. A proposed kinetic model of three-state transitions describes quantitatively the interstate conversion via reversible internal and external protonation. The excited neutral state fluorescence lifetime of EcGFP decays is significantly longer (i.e., larger fluorescence quantum yield) than most neutral GFPs, which can be exploited as a new spectral window in biological studies with multiple labels. The pH independence of the EcGFP neutral fluorescence lifetimes implies a ground-state interconversion mechanism for the observed fluorescence quenching in acidic environment.