Effects of threonine 203 replacements on excited-state dynamics and fluorescence properties of the green fluorescent protein (GFP)

Authors: Andreas D Kummer, Jens Wiehler, Hermann Rehaber, Christian Kompa, Boris Steipe, Maria Elisabeth Michel-Beyerle

DOI: 10.1021/jp9942522

Takeaways:

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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study of wild-type green fluorescent protein (GFP) and single-site mutants in which threonine at position 203 has been replaced by aliphatic and aromatic residues, i.e., by valine (V), isoleucine (I), phenylalanine (F), tyrosine (Y), and histidine (H).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 RHis 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 RHand 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.

Main

Owing to the missing hydroxy group of T203, there is less stabilization of R- and the corresponding absorption band is almost entirely lost.

Furthermore, the presence of threonine at position 203 is not required for ESPT to occur.