FIGURE 6.

Spectral properties of GFP variants before and after photoconversion. (a) Absorption spectra of the T203V mutant. The original absorption spectrum (black line) is only slightly affected by illumination with near-UV light (350–450 nm) from the arc lamp for 1 h (shaded line). Photoconversion is induced with an additional 2 min of illumination with the integral light of the arc lamp (dotted line). (b) Absorption spectra of S65G/T203Y before (solid line) and after (dotted line) photoconversion. Reversed photoconversion of the deprotonated chromophore form is observed with UV illumination (2 min with the integral arc lamp emission). S65G and S65G/T203V behave similarily (data not shown). (c) Comparison between the fluorescence lifetime of the deprotonated chromophore form in S65G/T203V before (black line) and after (shaded line) photoconversion. Excitation and detection wavelengths were chosen as λ_exc = 465–470 nm and λ_det = 515 nm, respectively. The faster fluorescence decay indicates a higher contribution of internal conversion in the photoproducts (cf. Table 4).