Unveiling How an Archetypal Fluorescent Protein Operates: Theoretical Perspective on the Ultrafast Excited State Dynamics of GFP Variant S65T/H148D

Abstract

Green fluorescent protein variant S65T/H148D has been reported to host a photocycle involving the photoinduced proton transfer reaction between the chromophore and residue Asp148 under 50 fs and without a measurable kinetic isotope effect, and experimental evidence is suggestive of the existence of a highly delocalized proton between these residues. The blinding speed at which this biological system undergoes proton transfer has been ascribed to the extreme increase of acidity of the GFP chromophore in the electronic excited state where proton transfer takes place. This work strives to present a coherent, complete, and balanced description of the dynamics of this specific variant of GFP in which it will be shown that this increase of acidity is insufficient to explain the behavior observed. This study tracks the behavior of this photosystem to the delicate interplay between structure and dynamics shown in the presence of solvent. In this way, it has been found that the dynamics of this protein intertwines its structure with the intervening solvent to give rise to effectively degenerate situations in what concerns the reactants and products of the proton transfer reaction in ground and, most importantly, photoexcited state, in terms of potential energy profiles associated with the proton migration. Under these conditions, proton transfer can occur in accordance with the experimental data available. This set of characteristics is possibly common to a host of other proton transfer based fluorescent proteins, and helps promoting GFP S65T/H148D to a case of archetypal significance. Thus, our results can be useful to understand the way many fluorescent proteins work and, more generally, the molecular basis for proton transfer reactions in proteins.

New insights into the structure–spectrum relationship in S65T/H148D and E222Q/H148D green fluorescent protein mutants: a theoretical assessment

“…38,39 In order to obtain a relatively long time of QM/MM simulation, we used an approach previously used by us with notable success. 21 In short, this approach requires the computation of a precursor trajectory from which to sample the starting configurations for several independent trajectories on which averaging is to be performed. The protocol followed was the same as that in our previous study, allowing us to simulate 1.0 ns at 300 K. In the case of E222Q/H148D a previous extra QM/MM equilibration of 40 ps was performed to ensure that the mutations manually introduced were accommodated.…”

Complete Proton Transfer Cycle in GFP and Its T203V and S205V Mutants

“…Particularly informative were mutations in the vicinity of the proton wire (Figure 1). Both the S65T mutation and replacement of E222 disrupt the proton transfer chain, so no I* emission occurs on excitation of A; however, a further mutation (H148D) rewires proton transfer, via a short low‐barrier H‐bond to D148, to which ESPT occurs on a sub‐picosecond time scale 2a. 7 In contrast, mutations T203V and T203Y shift the emission of I*, but do not significantly alter ESPT.…”

Ultrafast action chemistry in slow motion: atomistic description of the excitation and fluorescence processes in an archetypal fluorescent protein

“…All PE profiles were close to isoergic. However, no evidence could be found for the postulated extreme drop in pK a * value of Tyr66, 26 which had originally been derived from a theoretical study of the chromophore in solution and in a conformation in which both rings were co-planar. The situation was found to be different in the protein: accounting for the effects of the protein surroundings led to an estimate of the pK a * decrease that was much smaller.…”

“…[22][23][24] In this spirit, some of us performed a detailed study of the ground state of GFP S65T/H148D with the goal of explaining the reasons behind this ultrafast process. 25,26 Using quantum mechanical/ molecular mechanical (QM/MM) and molecular dynamics (MD) methods the geometry of the Cro-Asp148 unit was satisfactorily reproduced. 25 Moreover, QM potential energy (PE) profiles for proton transfer in the ground and excited electronic state of several snapshots from these simulations revealed that the proton transfer becomes more favored upon photoexcitation.…”