The three amino acids S65, T203, and E222 crucially determine the photophysical behavior of wild-type green fluorescent protein. We investigate the impact of four point mutations at these positions and their respective combinations on green fluorescent protein's photophysics using absorption spectroscopy, as well as steady-state and time-resolved fluorescence spectroscopy. Our results highlight the influence of the protein's hydrogen-bonding network on the equilibrium between the different chromophore states and on the efficiency of the excited-state proton transfer. The mutagenic approach allows us to separate different mechanisms responsible for fluorescence quenching, some of which were previously discussed theoretically. Our results will be useful for the development of new strategies for the generation of autofluorescent proteins with specific photophysical properties. One example presented here is a variant exhibiting uncommon blue fluorescence.
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study of wild-type green fluorescent protein (GFP) and single-site mutants in which threonine at position 203 has been replaced by aliphatic and aromatic residues, i.e., by valine (V), isoleucine (I), phenylalanine (F), tyrosine (Y), and histidine (H).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.
The optical spectra of the Aequorea victoria green fluorescent protein (GFP) are governed by an equilibrium between three different chromophore states. Mutants that predominantly show either the protonated (A) or the deprotonated (B) form of the chromophore have previously been described. In contrast, the I form, which is formed by rapid excited-state deprotonation of the A form of the chromophore, has only been described as an obligatory photochemical intermediate. We report the design of a new GFP mutant with a stabilized I form. For this purpose, we introduced two isosteric point mutations, Thr203Val and Glu222Gln, that selectively raise the potential energy of both the A and the B form. Knowledge of the absorption spectrum of the I form at room temperature allows the detailed analysis of concentration dependent changes in bulk wild-type(wt)-GFP spectra, as well as the determination of the dimerization constant of GFP. This information expands the use of GFP to that of a spectral probe for protein concentration. We determined energy differences between the chromophore ground states in the monomer and the dimer and reconstructed part of the potential energy surface.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.