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

Abstract

We have investigated the pH dependence of the dynamics of conformational fluctuations of green fluorescent protein mutants EGFP (F64L/S65T) and GFP-S65T in small ensembles of molecules in solution by using fluorescence correlation spectroscopy (FCS). FCS utilizes time-resolved measurements of fluctuations in the molecular fluorescence emission for determination of the intrinsic dynamics and thermodynamics of all processes that affect the fluorescence. Fluorescence excitation of a bulk solution of EGFP decreases to zero at low pH (pKa = 5.8) paralleled by a decrease of the absorption at 488 nm and an increase at 400 nm. Protonation of the hydroxyl group of Tyr-66, which is part of the chromophore, induces these changes. When FCS is used the fluctuations in the protonation state of the chromophore are time resolved. The autocorrelation function of fluorescence emission shows contributions from two chemical relaxation processes as well as diffusional concentration fluctuations. The time constant of the fast, pH-dependent chemical process decreases with pH from 300 microseconds at pH 7 to 45 microseconds at pH 5, while the time-average fraction of molecules in a nonfluorescent state increases to 80% in the same range. A second, pH-independent, process with a time constant of 340 microseconds and an associated fraction of 13% nonfluorescent molecules is observed between pH 8 and 11, possibly representing an internal proton transfer process and associated conformational rearrangements. The FCS data provide direct measures of the dynamics and the equilibrium properties of the protonation processes. Thus FCS is a convenient, intrinsically calibrated method for pH measurements in subfemtoliter volumes with nanomolar concentrations of EGFP.

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.

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.

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.

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.

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.

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.

Figure 7

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