Transient low-barrier hydrogen bond in the photoactive state of green fluorescent protein

Abstract

In this paper, we have analyzed the feasibility of spontaneous proton transfer in GFP at the Franck-Condon region directly after photoexcitation. Computation of a sizeable portion of the potential energy surface at the Franck-Condon region of A the structure shows the process of proton transfer to be unfavorable by 3 kcal mol(-1) in S1 if no further structural relaxation is permitted. The ground vibrational state is found to lie above the potential energy barrier of the proton transfer in both S0 and S1. Expectation values of the geometry reveal that the proton shared between the chromophore and W22, and the proton shared between Ser205 and Glu222 are very close to the center of the respective hydrogen bonds, giving support to the claim that the first transient intermediate detected after photoexcitation (I0*) has characteristics similar to those of a low-barrier hydrogen bond [Di Donato et al., Phys. Chem. Chem. Phys., 2012, 13, 16295]. A quantum dynamical calculation of the evolution in the excited state shows an even larger probability of finding those two protons close to the center compared to in the ground state, but no formation of the proton-transferred product is observed. A QM/MM photoactive state geometry optimization, initiated using a configuration obtained by taking the A minimum and moving the protons to the product side, yields a minimum energy structure with the protons transferred and in which the His148 residue is substantially closer to the now anionic chromophore. These results indicate that: (1) proton transfer is not possible if structural relaxation of the surroundings of the chromophore is prevented; (2) protons H1 and H3 especially are found very close to the point halfway between the donor and acceptor after photoexcitation when the zero-point energy is considered; (3) a geometrical parameter exists (the His148-Cro distance) under which the structure with the protons transferred is not a minimum, and that, if included, should lead to the fluorescing I* structure. The existence of an oscillating stationary state between the reactants and products of the triple proton transfer reaction can explain the dual emission reported for the I0* intermediate of wtGFP.

The mechanism of a green fluorescent protein proton shuttle unveiled in the time-resolved frequency domain by excited state ab initio dynamics

“…However, this hypothesis of causality between a photoactivated low frequency mode and the ESPT has been questioned in recent works. 26 , 33 …”

“…A more accurate description of the kinetics would have required the computation of a larger number of excited state trajectories and the use of high level wavefunction methods, 17 , 25 , 26 , 34 – 36 , 39 , 40 for example by adopting precomputed and high level parameterized potentials, and also including quantum effects. 25 , 26 , 39 , 40 However, these treatments are unfeasible when including dynamical effects of the chromophore and the entire protein, with no structural restraint in the excited state.…”

“… 37 , 38 Ab initio molecular dynamics (AIMD) studies have also been proposed on GFP models, 39 , 40 and on the entire GFP at the QM/MM level of theory, also including quantum effects. 26 In this latter study however, the modelling did not include the photoinduced structural relaxation and the process in the excited state remains endergonic. A recently calculated intrinsic reaction coordinate on a reduced GFP model did include the rearrangement of the chromophore pocket, although this provided the analysis of a limited region of the potential energy surface accessible to the system without fully accounting for the protein matrix effects.…”

“… 4 In contrast, the importance of modes involving heavy atoms did not emerge in other studies. 26 , 33 …”

Structure‐guided evolution of Green2 toward photostability and quantum yield enhancement by F145Y substitution

“…13,28 Engineering chromophore variants combined with simulation analysis demonstrated that appropriate perturbations to the chromophore or its environment lead to ESPT twisting to a more energetically favorable pathway, and molecular isomerization is more favorable after excitation; all of these result in a lower energy barrier between the ground and excited states. 13,29,30 Introducing the F145Y substitution into Green2 significantly lowered the transfer energy from the ground to the excited state, even in an acidic environment ( Figure 5). Accordingly, the QY of Green after introducing F145Y increased markedly, in line with previous results showing that QY of cyan FP with substitution of H148D is much higher than with two substitutions of Y145F and H148D.…”