Structural Basis of Fluorescence Fluctuation Dynamics of Green Fluorescent Proteins in Acidic Environments

Abstract

Green fluorescent proteins (GFP) have become powerful markers for numerous biological studies due to their robust fluorescence properties, site-specific labeling, pH-sensitivity, and mutations for multiple-site labeling. Fluorescence correlation spectroscopy (FCS) studies have indicated that fluorescence blinking of anionic GFP mutants take place on a time scale of 45-300 μs, depending on pH, and have been attributed to external proton transfer. Here we present experimental evidence indicating that conformational change of the protein β-barrel is a determining step for such external protonation of GFP-S65T (at low pH) using time-resolved fluorescence and polarization anisotropy measurements. While the average anionic fluorescence lifetime of GFP-S65T is reduced by ∼18% over pH 3.6-10.0 range, the fluorescence polarization anisotropy decays mostly as a singleexponential with a rotational time, φ=17±1 ns, that indicates an intact β-barrel with a hydrodynamic volume of 78±5 nm 3 . In contrast, the total fluorescence (525±50 nm) of the excited neutral state of S65T reveals a strong correlation between the fluorescence lifetime, structural conformation, and pH. The average fluorescence lifetime of the excited neutral state of S65T as a function of pH yields a pK a ≈5.9 in agreement with literature values using steady-state techniques. In contrast to the intact β-barrel at high pH, the anisotropy of neutral S65T (at pH≤pKa) decays as a biexponential (e.g., at pH 5.8, φ 1 =1.86 ns, β 1 =0.03, φ 2 =17.5 ns, and β 2 =0.25), which suggests a segmental mobility of the chromophore associated with conformational changes of the protein. The segmental motion of the S65T chromophore becomes faster with an enhanced amplitude ratio as pH is reduced. For comparative purposes, we also provide complementary FCS results on fluorescence blinking of the neutral-state of EGFP mutant (F64L/S65T), on a much slower time scale. Our results indicate that conformational rearrangement of the β-barrel and the amino acids surrounding the embedded chromophore is a rate determining step for external proton transfer and possibly cis/trans isomerization as non-radiative pathways that underlie fluorescence blinking of GFP mutants in acidic environment. In addition, the neutral-state transition is likely to be involved in the blinking process previously observed for the anionic-state transition in several GFP mutants.

Sensing of Intracellular Environments by Fluorescence Lifetime Imaging of Exogenous Fluorophores

“…86 A decrease in τave with lowering pH was also observed for the S65T variant in a buffer, when the neutral form was preferentially excited, and both fluorescence signals were detected. 87 It should be noted that the fluorophore structures of EGFP and the S65T variant are the same as that of wtGFP in Fig. 5.…”

“…The fluorescence lifetime of the anionic form is typically in the 1 -4 ns range, and that of the neutral form is in tens of picoseconds, or shorter. [86][87][88][89] Such a large difference in the fluorescence lifetime between these two forms can be applied to measure the medium pH by the lifetimebased ratiometric method. When both forms are simultaneously excited and the fluorescence from both of the forms is simultaneously detected, the fluorescence decay is a mixture of the decays of the neutral and anionic forms.…”

“…Then, the τave value depends on the population ratio between the neutral and anionic forms. [86][87][88][89] The pH dependence of τave was observed for enhanced GFP (EGFP: F64L/S65T), which has widely been used for imaging of biological systems. 86 It was reported that τave of EGFP becomes shorter with decreasing pH in a buffer solution, when the neutral form of the fluorophore is dominantly excited and the fluorescence from both the neutral and anionic forms is observed.…”

“…The fluorescence was detected at around 510 nm, where the fluorescence from both the neutral and anionic forms was observed. 86,87 It should be noted that the time-gated TD method was also adopted in the FLIM measurements, and the fluorescence lifetime was evaluated by assuming a single exponential decay. 30 It is shown in Fig.…”

“…86,87 The apparent pKa of EGFP in HeLa cell was then estimated to be 5.6 based on the results shown in Fig. 6.…”