Despite myriad challenges, clinicians see room for progress.
Image credit: Shutterstock/David Tadevosian.
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Contributed by Watt W. Webb
Article Figures & SI
Figures
- 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 pKa 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 kapp = kprot([H+] + [A−]) + kdeprot 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 τC 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.
Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy
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