No abstract

We report a comparative study of wild-type green fluorescent protein (GFP) and single-site mutants in which threonine at position 203 has been replaced by aliphatic and aromatic residues, i.e., by valine (V), isoleucine (I), phenylalanine (F), tyrosine (Y), and histidine (H). Steady-state absorption spectra reveal changes that reflect different charge distributions in the mutants as compared to wild-type GFP. While the absorption peak of the protonated fluorophore, RH, undergoes only a small red shift in all T203 mutants, a pronounced red shift is observed for the deprotonated form R-, ca. 1000 cm-1 for the aliphatic mutants T203V and T203I, ca. 1200 cm-1 for T203F, and 1360 cm-1 for T203Y. Thus, we conclude that a ground-state conformation higher in energy than the wild-type R- state is the predominant origin of the red shift in all the T203 mutants investigated. Furthermore, mutant-dependent changes in the ground-state equilibria of RH and R- result from at least two modes of electrostatic stabilization, one resting on hydrogen bonding as in T203 and the other one on π−π-stacking as in T203F and T203Y. Surprisingly, the deprotonation dynamics of RH* is only weakly affected by the mutations at position 203. Only in the most red-shifted mutant T203Y an additional ultrafast (1.7 ps) excited-state decay channel of RH* has been observed. The identical kinetics of both processes, decay of RH* and ground-state recovery of RH in T203Y, is discussed in terms of two mechanisms:  (i) rate-determining electron transfer from the protonated (or deprotonated) tyrosyl 203 residue to RH* followed by considerably faster recombination processes, which cannot occur in T203F for energetic reasons, and (ii) internal conversion in RH* favored by rotational motion around the exocyclic double bond.

Time-resolved fluorescence measurements at 275 K show that the excited-state lifetime of a model chromophore of the green fluorescent protein (GFP) substantially increases from subpicoseconds in low-viscosity solvents such as ethanol (η = 1.7 cP) to 30 ps in glycerol (η = 9.9 × 103 cP) and reaches 2.1 ns in glycerol glass at 150 K. At high temperatures the similarity of excited-state decay and ground-state recovery kinetics indicates internal conversion being responsible for the short fluorescence lifetimes. Their viscosity dependence reflects on a motion with a considerable amplitude that is damped by viscous drag and outweighs thermal activation as is concluded from measurements at different temperatures. In solution the neutral and the anionic forms of the model chromophore are similarly nonfluorescent in contrast to wild-type GFP and mutants where the deprotonated form is hardly undergoing internal conversion. Thus, the protein selectively restricts motional degrees of freedom of the chromophore in specific protonation states.