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.

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