Shedding light on the dark and weakly fluorescent states of green fluorescent proteins

Abstract

Recent experiments on various similar green fluorescent protein (GFP) mutants at the single-molecule level and in solution provide evidence of previously unknown short- and long-lived "dark" states and of related excited-state decay channels. Here, we present quantum chemical calculations on cis-trans photoisomerization paths of neutral, anionic, and zwitterionic GFP chromophores in their ground and first singlet excited states that explain the observed behaviors from a common perspective. The results suggest that favorable radiationless decay channels can exist for the different protonation states along these isomerizations, which apparently proceed via conical intersections. These channels are suggested to rationalize the observed dramatic reduction of fluorescence in solution. The observed single-molecule fast blinking is attributed to conversions between the fluorescent anionic and the dark zwitterionic forms whereas slow switching is attributed to conversions between the anionic and the neutral forms. The predicted nonadiabatic crossings are seen to rationalize the origins of a variety of experimental observations on a common basis and may have broad implications for photobiophysical mechanisms in GFP.

Figure 1

Models of p-hydroxybenzylideneimidazolidinone GFP chromophore in the neutral, anionic, and zwitterionic forms used in the quantum chemical calculations, shown in those resonance structures that best represent the calculated bond orders. The covalent links with the Ser65 and Gly67 C_α atoms are replaced by hydrogen atoms (indicated as H′). The torsional degrees of freedom around the two ring-bridging bonds considered here are denoted by ϕ, τ (C—C—C—N), and HT (hula-twist (27), concerted and simultaneous motion around ϕ and τ), respectively. Rotation by 180° around ϕ leaves the structure unchanged. The configurations displayed represent τ = 0° and are referred to as cis configurations. The upper panels show OM2 (ref. ; W.W. and W. Thiel, unpublished work)/PERTCI (29) energy profiles for rotation around the dihedral angles τ and ϕ and for the HT motion in the ground and first singlet excited states. Calculated values are marked by dots, and the Gaussian profiles are shown as visual aid. Note that, for these calculations, we fixed the dihedral angles while relaxing all other degrees of freedom.

Figure 2

Model for the photophysics of GFP as inferred from our quantum chemical calculations. Excited states are labeled by asterisks. Note that barriers may exist for processes of types 2 and 3. Excitation arrows are omitted for simplicity. The relative free energies of ground state forms A, B, I, and Z depend on the protein environment and, thus, on the specific mutant.

Figure 3

Superposition of three structures of wild-type GFP generated by molecular dynamics simulations. The dotted structure is a configuration from a previous MD simulation of GFP with a cis chromophore after 300 ps of equilibration (39). This structure was used to model a trans chromophore by rotation around τ by 180° (gray structure); 465 ps of simulation were performed starting from this configuration (32), and the structure in black is an average structure over the last 80 ps. Cis and trans configuration of the chromophore overlap well, but the trans chromophore is less well coordinated by surrounding protein residues.