Figure 1.
(A) Chromophore (MYG) in the trans conformation and adjacent amino acid side chains. Ser158, Glu215, and the crystallographic water molecule W233 are hydrogen-bonded to MYG (dashed lines). His197 is π-stacked to the MYG phenoxy-moiety and forms a hydrogen bond to Glu215 (dashed line). The carbon skeleton of the quantum mechanical (QM) subsystem is shown in cyan, and the carbon atoms modelled by molecular mechanics (MM) are shown in orange. (B) Schematic drawings of the different MYG protonation states considered in this work.
Figure 2.
Schematic representation of potential energy surfaces.
The excited (S1, red) and ground (S0, green) states of the neutral chromophore are shown along the trans-cis isomerization coordinate (torsion A) and a skeletal deformation coordinate of the chromophore. Radiationless decay occurs at the S1/S0 conical intersection (CI, dashed white line). In this representation, the CI occurs as an extended seam, because the torsion coordinate is from the (N-2)-dimensional intersection space, and the skeletal deformation coordinate is from the 2-dimensional branching pace. The dashed yellow line represents the path sampled in a QM/MM photoisomerization trajectory.
Table 1.
Excited state lifetimes and final conformations from the MD simulations initiated in the neutral trans chromophore conformation.
Figure 3.
Trans-cis isomerization of the neutral chromophore.
(A) Chromophore (MYG) conical intersection geometry adopted during the MD simulation. MYG forms hydrogen bonds to Arg92, Glu145, Ser158, and Glu215. Color code as in Figure 1. (B) Ground (S0, black) and excited (S1, red) potential energy traces along the QM/MM molecular dynamics trajectory. Photon absorption (blue arrow) excites the chromophore into S1 (yellow area) until it decays back to S0 at the conical intersection seam (dashed line). (C) Time-evolution of the ring-bridging torsion angles A (magenta) and B (blue). (D, E) Change of the hydrogen bonding network in the chromophore cavity during trans-cis isomerization. The MYG-Arg92 (black), MYG-Glu145 (green), MYG-Ser158 (red), Lys67-Glu195 (cyan, residues not shown in (A)), and Lys67-Glu145 (green) hydrogen bonds were stable during isomerization. Additional hydrogen bonds between MYG and Glu215 (blue) as well as between His197 and Glu215 (orange) were transiently formed.
Table 2.
Excited state lifetimes and final conformations from the MD simulations initiated in the neutral cis chromophore conformation.
Figure 4.
Influence of the protein environment on the photoisomerization process of the neutral asFP595 chromophore.
(A, C) Ground and excited state energies along trans-to-cis (A) and cis-to-trans (C) isomerization trajectories (run b, Table 1 and run a, Table 2). The protein environment stabilizes S0 and S1 (black and red lines, respectively) relative to the gas phase (dashed blue and green lines, respectively). (B, D) Energy difference between the protein and the gas phase. ΔE(S0) = E(S0, protein)−E(S0, gas phase) is plotted in black, ΔE(S1) = E(S1, protein)−E(S1, gas phase) in red. The protein environment energetically stabilizes S1 more strongly than S0. The vertical dashed black line represents the surface crossing. The energy offset in (A) and (C) is 1.9699×106 kJ/mol.
Figure 5.
Ultra-fast internal conversion mechanism of the trans anion.
(A) Snapshot at the conical intersection: the chromophore is twisted around torsion B, yet the hydrogen bonded network in the chromophore cavity remains intact. (B) Ground (S0, black) and excited (S1, red) potential energy traces along the QM/MM molecular dynamics trajectory. Photon absorption (green arrow) brings the chromophore into S1 (yellow area) until it decays back to S0 at the conical intersection seam (dashed line). (C) Time-evolution of the torsion angles A (magenta) and B (blue). (D) S0 and S1 energies along a representative excited state trajectory of Atrans. The protein environment strongly stabilizes S0 and S1 (black and red lines, respectively) relative to the gas phase (dashed blue and green lines, respectively). The energy offset is 1.9686×106 kJ/mol. (E) Energy difference ΔE between the protein and the gas phase for S0 (black) and S1 (red).
Figure 6.
Hydrogen bonding network in the chromophore cavity during force field simulations of zwitterionic chromophores.
(A, C) Snapshots from MD simulations of Ztrans and Zcis, respectively. The blue dashed lines indicates the distance between the NH proton of MYG and Glu215, and the red dashed line that between the OH-group of Glu215 and the Nδ atom of His197. (B, D) Time-evolution of the two hydrogen bonds shown in (A) and (C) during representative force field MD simulations.
Figure 7.
Scheme of the proposed decarboxylation of Glu215, which yields an irreversibly fluorescent zwitterion.
Figure 8.
Scheme of the reversible photoswitching mechanism of asFP595 proposed in this work.
The fluorescent state Zcis is highlighted. The green arrows indicate ground state equilibria, whereas the red arrows indicate excited state processes. The major protonation states are the zwitterionic and the anionic chromophores in the trans conformation, and the neutral chromophore in the cis conformation, as indicated in the square brackets.