PMC

FCS measurements of Citrine, as a function of pH, reveal an external protonation process. (a) The correlation functions, (b) rate constants (■) with the best fit (dotted line; see text), and (c) fractions (□) for proton binding. (b) The pH-independent photoconversion rate τ₁⁻¹ =1.6 ± 0.4 × 10³⋅s⁻¹ (○) and (c) dark fraction (●) f₁ = 0.30 ± 0.06 are superimposed.

Time-resolved fluorescence anisotropy of dsRed and Citrine (pH 9.0) by using TCSPC. The anisotropy of 490-nm excited Citrine decays as a single exponential with rotational time φ = 16 ± 2 ns. To the contrary, dsRed anisotropy decays as a biexponential under the same experimental conditions, with a fast φ_f = 211 ps (indicated by ↑) and a slow φ_S = 53 ± 8 ns rotational time with fast-to-slow amplitude ratio of ≈0.13.

Absorption and emission spectra of dsRed and Citrine. (a) Normalized one-photon absorption (solid line and points) and emission (open points) spectra of dsRed (●○) and Citrine (▴▾▵▿), at pH 9. A new absorption band (▾) of Citrine appears (412 nm) in pH 4.9 buffer whereas its emission (▿) reveals a broader blue wing of the 524-nm band. The nonnormalized 514-nm absorption band of Citrine in pH 9 is ≈3.7 times weaker in pH 4.9 before normalization. (b) The 2P-excitation cross section of dsRed (●) and Citrine (○) over the 730–990 nm, where GM = 10⁻⁵⁰ cm⁴⋅s/photon. Note that σ_2P of dsRed continues to rise at 990 nm.

Fluorescence correlation spectra of dsRed. (a) Excitation intensity dependence of photoconversion kinetics of dsRed (pH 9.0) FCS correlation curves as a function of k_ex at 488 nm (≈0.4–7.3 kW/cm²). (b) Light-driven fluorescence flicker fraction and (c) rates of dsRed. The dark fraction (f₁ = 0.30 ± 0.04) appears constant below saturation (with a minor decline at low k_ex and the rate depends linearly on intensity with a slope of (2.9 ± 0.2) × 10⁻³ and an intercept of 400 ± 30 Hz as k_ex→0. Autocorrelation spectra of dsRed (a) at low k_ex show lack of pH dependence in the pH range 3.9 to 11. (d) The fluorescence flicker rate (1.7 ± 0.1 × 10³ s⁻¹) and dark fraction (0.41 ± 0.03) are clearly independent of pH.

First excited electronic-state fluorescence decays of dsRed and Citrine by using TCSPC. First excited anionic (S₁^A, λ_ex = 490 nm), and neutral (S₁^N, λ_ex = 405 nm) state dynamics of dsRed and Citrine. After 490-nm excitation of dsRed (pH 9), the fluorescence decays as a single exponential with τ_f = 3.67 ns and χ² = 1.08 (curve 1), whereas τ_f = 3.61 ns and χ² = 1.06 (curve 1) for Citrine. Unlike dsRed, Citrine fluorescence exhibits a biexponential decay (curve 2: τ_f1 = 3.31 ns, τ_f2 = 880 ps, amplitude ratio a₂/a₁≈0.26, and χ² = 1.06) at pH 4.9. The fluorescence decays (curves 3–5), following the S₀^N→S₁^N transition in Citrine (pH 4.9) by using 405 nm, are also shown as function of detection wavelength: λ_f = 460 nm (curve 5: τ_f1 = 24 ps, τ_f2 = 268 ps, τ_f3 = 2.11 ns, amplitude ratios a₂/a₁≈0.11, a₃/a₁≈0.04, and χ² = 1.06); λ_f = 500 nm (curve 4: τ_f1 = 51 ps, τ_f2 = 452 ps, τ_f3 = 2.63 ns, a₂/a₁≈0.16, a₃/a₁≈0.06, and χ² = 1.21); and λ_f = 520 nm (curve 3: λ_f1 = 54 ps, λ_f2 = 712 ps, λ_f3 = 3.12 ns, a₂/a₁≈0.19, a₃/a₁≈0.16, and χ² = 1.28).

Schematic PES of the anionic (S₀^A→S₁^A) and neutral (S₀^N→S₁^N) and intermediate (S₀^I→S₁^I) state transitions in Citrine. The reaction coordinate is presumably internal proton transfer between the chromophore, particularly Y66, and the immediate hydrogen bond network (). The crossing point between PESs along the reaction coordinate forms a barrier whose height and width depend on pH and mutation type/site. The anionic S₁^A state of Citrine decays via fluorescence at a rate τ_f⁻¹ and, consequently, a range of vibrational levels on the S₀^A-PES will be populated followed by a probability for nonradiative vibrational relaxation to the bottom of the S₀^A, S₀^I, and S₀^N PESs depending on the barriers. Fluorescence decays of the neutral S₁^N state of Citrine chromophore suggest an efficient nonradiative channel (e.g., intramolecular excited-state proton transfer) that competes with fluorescence. It is most likely that the S₁ state in dsRed has anionic character analogous to most GFP mutants (, ), and the neutral state S₀^N→S₁^N transition probability is negligible.