Structure and mechanism of the reversible photoswitch of a fluorescent protein

Abstract

Proteins that can be reversibly photoswitched between a fluorescent and a nonfluorescent state bear enormous potential in diverse fields, such as data storage, in vivo protein tracking, and subdiffraction resolution light microscopy. However, these proteins could hitherto not live up to their full potential because the molecular switching mechanism is not resolved. Here, we clarify the molecular photoswitching mechanism of asFP595, a green fluorescent protein (GFP)-like protein that can be transferred from a nonfluorescent "off" to a fluorescent "on" state and back again, by green and blue light, respectively. To this end, we establish reversible photoswitching of fluorescence in whole protein crystals and show that the switching kinetics in the crystal is identical with that in solution. Subsequent x-ray analysis demonstrated that upon the absorption of a green photon, the chromophore isomerizes from a trans (off) to a cis (on) state. Molecular dynamics calculations suggest that isomerization occurs through a bottom hula twist mechanism with concomitant rotation of both bonds of the chromophoric methine ring bridge. This insight into the switching mechanism should facilitate the targeted design of photoswitchable proteins. Reversible photoswitching of the protein chromophore system within intact crystals also constitutes a step toward the use of fluorescent proteins in three-dimensional data recording.

Fig. 1.

Overall structure of asFP595. A schematic ribbon representation of the quaternary tetrameric structure of asFP595 shows the four molecules in different colors and the chromophores highlighted in red.

Fig. 2.

Reversible photoswitching of a protein crystal under a fluorescence microscope. (a) asFP595-A143S crystals in the equilibrium state are barely fluorescent. Irradiation with green light (550 ± 20 nm, 50 W·cm^–2, 170 sec) yields brightly fluorescent crystals. The fluorescence is quenched by gentle irradiation with blue light (450 ± 20 nm, 3 W·cm^–2, 24 sec); the quenching also takes place while simultaneously irradiating with green light (required to survey the fluorescence). (Scale bar: 20 μm.) (b) Brightfield image of the asFP595-A143S crystal. (c)On/off cycles of the asFP595-A143S crystal fluorescence by using the same intensities as in a. Excitation sequence: green light, 170 sec; green plus blue light, 24 sec; blue light, 1 sec. Fluorescence was recorded during the first and the second section of the sequence.

Fig. 3.

Switching kinetics of asFP595-A143S are the same in solution and in crystals. asFP595-A143S crystals (dotted) and asFP595-A143S in solution (solid) were fully switched on (a) or off (b) by green light or by blue light, respectively. Shown is the subsequent return of the fluorescence to the equilibrium level.

Fig. 4.

Trans and cis conformation of the MYG chromophore in the off and the on state. Views of the MYG chromophores after least-squares superpositioning of all Cα atoms. (a) MYG chromophores of asFP595 variants as indicated in the figure. Atoms are color-coded by atom type (carbon wt asFP595, cyan; carbon asFP595-A143S, salmon; carbon asFP595-S158V, beige; oxygen, red; nitrogen, blue). Final 2F_o – F_c electron densities around the chromophores are contoured at the 1σ level. For asFP595-A143S (irradiated for 1 min) both isomerization states are depicted, because both structures were observed in a single crystal with equal proportions. (b) Overlay of the four chromophore structures. Color-coding as in a.

Fig. 5.

MD simulations of the switching mechanism in asFP595. (a) The two possible isomerization mechanisms. (b) Forces due to the protein matrix opposing chromophore isomerization were calculated by nonequilibrium force-probe MD simulations; 10 trajectories were averaged for the four possible pathways (solid lines). For the rotate mechanism, the MYG p-hydroxyphenyl ring can either rotate toward the initially coplanar H197 (R^top) or toward the other side (R^bot). Likewise, during HT isomerization, the bridging methine group can move along a top (HT^top) or bottom (HT^bot) pathway. Control simulations of the chromophore in water show similar forces for the R and HT mechanisms (dashed curves). (Inset) MYG chromophore and relevant torsion angles. (c) Spontaneous trans-cis isomerization during free excited state MD simulations for the chromophore within the protein matrix (Upper) and in water (Lower), monitored through the dihedral angles τ (dashed curves) and ϕ (solid curves). The protein favors the HT^bot mechanism, with both dihedral angles rotating simultaneously within a narrow time frame. Simulation of protein-free isomerization of the chromophore in water also follows HT; both directions (HT^top and HT^bot) are observed in this case.