Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy

pH Dependence of Fluorescence and Absorption.

Fluorescence emission of GFP is very pH sensitive, dropping to zero at very low and high pH values (11, 12, 28, 29). The emission intensity of EGFP as a function of pH is shown in the Inset of Fig. 1. The emission maximum lies at 509 nm with a pronounced shoulder at about 540 nm. Shapes and maxima of the emission spectra are essentially pH independent. A fit of the emission intensity F versus pH according to the equation F = x + y/(1 + 10^z), where z is (pK_a − pH) and x and y are related to the offset and dynamic range of the data, yields a pK_a of 5.8 ± 0.1, indicating a single protonation step to be primarily responsible for the decreased fluorescence. From the equation Δ_rG° = −RTlnK the standard free reaction energy may be calculated to be about 33 ± 1 kJ/mol at room temperature. At pH > 12 there is a drastic loss of fluorescence. Interestingly, fluorescence of the mutant GFP-Y66W first increases by a factor of 2 going from pH 8 to 5.5 but then also decays to zero at even lower pH (data not shown).

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Figure 1

Fluorescence excitation spectra of EGFP in 100 mM potassium phosphate/10 mM citrate at various pH values (down from the top): 9.0, 8.0, 11.0, 7.0, 6.5, 6.0, 5.5, and 5.0. (Inset) Fluorescence emission intensity at 510 nm (■), fluorescence excitation intensity at 490 nm (•), and absorption at 488 nm (♦) versus pH. The solid line is fit to the emission intensity from pH 4.0 to 9.0 yielding a pK_a of 5.8 ± 0.1.

Excitation spectra of EGFP peak at 488 nm with a shoulder at about 465 nm and an additional ≈13% contribution at 400 nm. The pH dependence of the spectra is shown in Fig. 1. The shapes of the spectra at pH 9.0 and 5.0 are very similar except for a slightly higher excitation in the range of 400 nm at pH 9.0. Intensity of excitation versus pH (Fig. 1 Inset) is in good agreement with that of emission with a fitted pK_a of 5.9 ± 0.1.

Like emission and excitation, absorption of EGFP is strongly pH dependent (Fig. 2). At pH 9.0 absorption and excitation spectra are basically congruent. Lowering the pH leads to an absorption decrease at 488 nm and a concomitant increase at 398 nm with a clear isosbestic point (425 nm), as one would expect for a single protonation reaction. Absorption at 488 nm versus pH is included in the Inset of Fig. 1 and shows the same behavior as fluorescence emission and excitation. This behavior implies that the reduced emission at low pH is due to a decreased molar absorption coefficient rather than a lower quantum efficiency. Given a pK_a of 5.8 for the protonation equilibrium, one can calculate the molar absorption coefficient at 398 nm of the low-pH species to be about 38% of the high-pH species at 488 nm. Since the excitation spectra at low pH do not show an increased contribution around 400 nm, the species produced at low pH does not contribute to the fluorescence around 500 nm.

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Figure 2

Absorption spectra of EGFP in 100 mM potassium phosphate/10 mM citrate at various pH values (down from the top at 490 nm): 10.0, 9.0. 8.0. 7.0. 6.5, 6.0, 5.5, and 5.0.

FCS.

In Fig. 3 autocorrelations (blue curves) of EGFP at pH values from 5.0 to 11.0 are presented. The autocorrelation function G(t) is defined as G(t) = 〈δF(t)δF(0)〉/〈F(t)〉², where F(t) is the fluorescence obtained from the volume at time t, angled brackets denote time averages, and δF(t) = F(t) − 〈F(t)〉. Above pH 8.0 the curves are basically indistinguishable. With increasingly acidic pH a new fast component grows with increasing amplitude and decreasing time constant; this might represent the expected protonation fluctuation of the chromophore. The correlation shoulder in the millisecond time range is known to be due to diffusional fluctuations of the number of EGFP molecules in the optically established focal volume comprising the grand canonical ensemble. The correlation function describing the fluctuations around equilibrium of a simple AH ⇌ A⁻ + H⁺ reaction coupled with diffusion is given by 1 where N is the average number of molecules in the probe volume, F is the average fraction of molecules in the nonfluorescent state (AH), ω describes the length-to-diameter ratio of the three-dimensional gaussian volume element, and τ_C and τ_D are the chemical and diffusional relaxation times, respectively.

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Figure 3

Autocorrelations G(t) for EGFP (blue curves) at different pH values (normalized to 1 at 10 μs). Fits of Eq. 2 to the data are shown as dotted lines. The decay of G(t) at high pH is dominated by diffusional relaxation, whereas upon decreasing pH, chemical relaxation at shorter time is growing in amplitude and speed. (Inset) Autocorrelations of GFP-Y66W (red curves) at pH 9.0 (solid line) and 5.5 (broken line) showing absence of pH-dependent fast fluorescence flicker in this mutant, which lacks a protonatable hydroxyl on the chromophore.

Fitting Eq. 1 to the data yields a decreasing τ_C (from 450 ± 65 μs to 50 ± 4 μs) and an increasing fraction F (up to 80% at pH 5.0) with decreasing pH. However, from pH 8.0 to 11.0 there is a reproducible, pH-independent, dark fraction of 13% ± 2% with an associated time constant of ≈450 μs. Finding this constant fraction over a wide pH range indicates that AH ⇌ A⁻ + H⁺ does not describe the system completely. To account for the high-pH fraction we invoke a pH-independent internal protonation process—i.e., the proton fluctuates between the chromophore hydroxyl group and an internal proton binding site B⁻, as has been suggested on the basis of structural studies (6, 7). At low pH a second proton from the bulk solution can protonate the chromophore as hypothesized above, inducing a nonfluorescent state:

Species A⁻ is the only one contributing to fluorescence. Assuming for simplicity that protonation from the solution is substantially faster than the internal process (τ_C < τ′_C) and neglecting reactions between species AH and AH′ (molar ratio of A⁻ to AH′ ≈ 8:1), the autocorrelation function is approximated by 2 where G_diff(t) = [(1 + t/τ_D)(1 + t/ω²τ_D)^0.5]⁻¹ describes the diffusional part, the pre-exponential factors are P = [K[H⁺]{1 + K′(1 + K[H⁺])}]/[K′(1 + K[H⁺])] and P′ = 1/[K′(1 + K[H⁺])], and [H⁺] denotes the hydrogen ion concentration. The equilibrium constants are K = [AH]/[A⁻][H⁺] and K′ = [A⁻]/[AH′], and the time constants τ_C and τ′_C are associated with external and internal protonation, respectively. Between pH 9 and 11 Eq. 2 was fitted to the data, using the approximation P ≈ 0; P′ ≈ 1/K′. This yields an average diffusion time τ_D of 4.2 ± 0.3 ms, a K′ of 7.55 ± 0.60 corresponding to a 12% ± 1% fraction of nonfluorescent molecules, and τ_C′ = 341 ± 48 μs, which is somewhat faster than the results obtained with the single-exponential fit (450 μs). Data taken at 1/3 the usual excitation intensity show a 30% increase in both K′ and τ′_C, suggesting the possible involvement of photoinduced processes at high pH.

The results of fitting the low-pH data with K′ and τ′_C fixed at the average values (above) are shown in Fig. 4. As expected, the apparent rate constant k_app (= 1/τ_C) increases with decreasing pH. However, the equilibrium constant K appears to increase by a factor of 2.8 from pH 5.0 to 7.0, which may be due to the approximation τ_C ≪ τ′_C ≪ τ_D assumed in the autocorrelation function 2. The rate constants for protonation and deprotonation at low pH can be obtained from a fit of k_app = 1/τ_C = k_prot([H⁺] + [A⁻]) + k_deprot ≈ k_prot[H⁺] + k_deprot because the concentration of deprotonated protein [A⁻] (in the nanomolar range) is negligible compared with [H⁺] below pH 7.0 (Fig. 4). This fit yields k_prot = (1.53 ± 0.06) × 10⁹ M⁻¹⋅s⁻¹ and k_deprot = (4.48 ± 0.27) × 10³ s⁻¹, from which a pK_a [= log(k_prot/k_deprot)] of 5.5 ± 0.3 can be calculated, in close agreement within the uncertainties to the value obtained from bulk measurements (see above). The mutant S65T behaves very similarly, although the chemical relaxation at low pH is about twice as fast as for EGFP (Fig. 4). The same analysis yields k_prot = (3.45 ± 0.32) × 10⁹ M⁻¹⋅s⁻¹, k_deprot = (9.05 ± 1.52) × 10³ s⁻¹, and a pK_a of 5.6 ± 0.4 for GFP-S65T.

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Figure 4

Results for the apparent rate constants of chemical relaxation as obtained from fits of the correlation curves shown in Fig. 3, using Eq. 2 with the high-pH approximation for pH 8 to 11. For pH 5.0 to 7.0 the values for K′ and τ′_C were fixed at the average values obtained from pH 8.0 to 11.0. Open symbols, GFP-S65T; closed symbols, EGFP. Solid lines, fits of the equation k_app = k_prot([H⁺] + [A⁻]) + k_deprot to the data points from pH 5.0 to 7.0.

Control experiments were performed to exclude other possible origins of the fast decay time of the correlation curve at low pH—e.g., a light-induced process such as triplet formation or diffusion of a small contaminating molecule. In case of triplet formation the associated time constant is expected to depend on light intensity. Measurements taken at 8- and 80-μW laser power (Fig. 5B) demonstrate that the time constant for the fast (protonation) process does not change within experimental error (208 ± 8 μs at 8 μW and 219 ± 5 μs at 80 μW), excluding a photoreaction for the low-pH process. The curve at higher power, however, shows an additional contribution at even shorter times (≈30 μs), which is entirely consistent with triplet formation but merits further investigation.

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Figure 5

Influence of light intensity and detection volume on time constants. (A) At pH 5.5 fiber diameters of 25, 50, and 100 μm (left to right) were used to vary spot size. Fits are shown as dotted lines. (B) Autocorrelation curves at 8 and 80 μW.

The possibility of a fast-diffusing contaminant can be checked by changing the focal volume size by using different fiber diameters of 25, 50, and 100 μm (Fig. 5A). Whereas the diffusion time τ_D increases by a factor of 3 as expected, again the chemical relaxation time τ_C is unchanged within experimental error (124 ± 2 μs, 121 ± 4 μs, and 130 ± 3 μs). This result excludes a diffusional relaxation or photobleaching for the fast process and confirms the assignment of the slow decay time (4.2 ms) to EGFP diffusion.

Most convincing evidence for a protonation fluctuation as the basis for the chemical kinetics is gained from measurements of the mutant Y66W, which does not posses a protonatable hydroxyl group on the chromophore. In Fig. 3 the red curves show autocorrelations obtained at pH 9.0 and 5.5. Evidently, there is no additional kinetic process at low pH as in the case of EGFP and GFP-S65T.

From the above results we conclude that the pH-dependent kinetic component at low pH is based on a protonation/deprotonation reaction of the chromophore. Since one might expect an isotope effect on the kinetics of proton movement we investigated EGFP in D₂O at different pD values. Although the scatter is much higher in the D₂O data, there seems to be an increase by a factor of 1.25 in τ′_C at high pH. This result might suggest that proton transfer is the rate-limiting step; however, D₂O also has a 1.23 times higher viscosity than does H₂O, which might influence kinetics.

To distinguish between viscosity and isotope effect we increased viscosity of the solution by adding glycerol. Diffusion time τ_D (pH 5.5 and pH 10) and the chemical relaxation kinetics τ_C at pH 5.5 as well as τ′_C at pH 10 increase linearly with viscosity by factors of approximately 6.6 ± 0.3, 0.11 ± 0.01, and 0.61 ± 0.14, respectively (data not shown). Correspondingly, diffusion in D₂O is slowed down by an expected factor of ≈1.25 due to the 1.23 times higher viscosity of D₂O compared with H₂O. This finding suggests that the increase in chemical relaxation times in D₂O is a viscosity rather than an isotope effect and that proton transfer is not limiting. The high-pH process was ascribed to an internal protonation fluctuation. If such a proton movement is coupled to protein conformational changes it would be expected that its kinetics is influenced by solvent viscosity.

The kinetics of protonation reactions might be expected to be influenced by buffer concentration and/or ionic strength of the solution. In Fig. 6 we show that the ionic strength does not change the kinetics of the low-pH protonation process at constant buffer concentration. Inversely, a strong decrease of τ_C is seen with increased buffer concentration at constant ionic strength. This result seems to implicate buffer molecules in proton transfer reactions into the EGFP protein.

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Figure 6

Effect of ionic strength on the chemical relaxation time τ_C at pH 5.5 with constant phosphate buffer concentration of 10 mM, and effect of phosphate buffer concentrations with the ionic strength kept constant by adding appropriate amounts of KCl.

To obtain more information on the thermodynamics of the protonation equilibrium we performed a series of experiments at pH 6.5 in the temperature range from 15°C to 50°C. The temperature dependence of the equilibrium constant K allows the calculation of the standard reaction enthalpy Δ_rH° according to the van’t Hoff equation dlnK/d(1/T) = −Δ_rH°/R. From the slope of a linear fit to the graph lnK versus 1/T (Fig. 7) we obtain Δ_rH° = −11.5 ± 0.7 kJ/mol, and using Δ_rG° = Δ_rH° − TΔ_rS° we obtain a standard reaction entropy at room temperature of Δ_rS° = −150 ± 7 J/mol⋅K.

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Figure 7

Temperature dependence of equilibrium constant K at pH 6.5. Dotted line, linear fit to the data points.