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

Abstract

The green fluorescent protein (GFP) variant S65T/H148D recovers the A-band fluorescence lost in the single mutant S65T, and it has been established that Asp148 is the alternate proton acceptor for the excited state proton transfer (ESPT). This mutant has been widely studied and presents unique spectroscopic properties, such as an ultrafast rise in the fluorescence (<50 fs). Also it exhibits a red-shift of the A absorption band of 20 nm with respect to wt-GFP's. The double mutant E222Q/H148D presents a very similar behaviour, at least within the experimental data available (which is scarcer than those of S65T/H148D). By means of dynamic theoretical studies we have been able to (1) reproduce and thoroughly analyse the red-shifted absorption spectra of both mutants and (2) predict the structure that the variant E222Q/H148D (for which there is no X-ray-resolved structure available) most probably adopts in water at room temperature. Our results deepen the understanding of the way GFP variants work and give some new insights into the rational design of fluorescent proteins and biological photosystems in general.

Chromophore interactions leading to different absorption spectra in mNeptune1 and mCardinal red fluorescent proteins

“…This behavior has been previously described in the chromophore of GFP and other fluorescent proteins and would serve to justify the idea that electron withdrawing groups placed appropriately close to the imidazolinone moiety can preferentially stabilize this excited state, bringing the excitation wavelength up, as suggested by Lin et al 15 In connection to this, we verified that the same effect could be obtained by the presence of a negative charge in the neighborhood of the tyrosine moiety in a computational study of GFP mutants S65T/H148D and E222Q/H148D. 46 The other two excited states which contribute to band II can be described as involving also the excitation between local p-orbitals. Even though the specific shape of the NTOs implied varies, the fact is that both excitations occur at very similar wavelengths and with commensurate intensities.…”

Cutting the Gordian knot of excited-state modeling in complex environments

“…In agreement with previous studies [12,19,39,118], we find that CAM-B3LYP gives blue-shifted values for the excitation energies of these photoactive systems. For the B form, the average excitation energy over 50 frames is 3.07 ± 0.01, which is almost 0.5 eV blue-shifted with respect to the experimental absorption maximum of 2.59 eV.…”

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

“…The two SOMOs are of p symmetry and are the main orbitals involved in the description of the photoactive excited state. 25 The other active-space orbitals are comprised of the three highest doubly occupied p orbitals and the five other highest occupied orbitals, as well as the three lowest unoccupied p orbitals and the five other lowest unoccupied orbitals. For the MRCI treatment, three configuration state functions were chosen as references, namely the SCF configuration and the two closed-shell configurations derived therefrom (i.e., all singlet configurations that can be generated from HOMO and LUMO of the closed-shell ground state).…”

“…S4 in the ESI †). 25 Hence, the HOMO-LUMO excitation breaks, or weakens significantly, the double bond between these atoms. As a consequence rotation around the C 6 C 4 bond becomes much easier than in the ground state.…”

“…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. All PE profiles were close to isoergic.…”