Structural basis for dual excitation and photoisomerization of the Aequorea victoria green fluorescent protein

Abstract

The 2.1-A resolution crystal structure of wild-type green fluorescent protein and comparison of it with the recently determined structure of the Ser-65 --> Thr (S65T) mutant explains the dual wavelength absorption and photoisomerization properties of the wild-type protein. The two absorption maxima are caused by a change in the ionization state of the chromophore. The equilibrium between these states appears to be governed by a hydrogen bond network that permits proton transfer between the chromophore and neighboring side chains. The predominant neutral form of the fluorophore maximally absorbs at 395 nm. It is maintained by the carboxylate of Glu-222 through electrostatic repulsion and hydrogen bonding via a bound water molecule and Ser-205. The ionized form of the fluorophore, absorbing at 475 nm, is present in a minor fraction of the native protein. Glu-222 donates its charge to the fluorophore by proton abstraction through a hydrogen bond network, involving Ser-205 and bound water. Further stabilization of the ionized state of the fluorophore occurs through a rearrangement of the side chains of Thr-203 and His-148. UV irradiation shifts the ratio of the two absorption maxima by pumping a proton relay from the neutral chromophore's excited state to Glu-222. Loss of the Ser-205-Glu-222 hydrogen bond and isomerization of neutral Glu-222 explains the slow return to the equilibrium dark-adapted state of the chromophore. In the S65T structure, steric hindrance by the extra methyl group stabilizes a hydrogen bonding network, which prevents ionization of Glu-222. Therefore the fluorophore is permanently ionized, causing only a 489-nm excitation peak. This new understanding of proton redistribution in green fluorescent protein should enable engineering of environmentally sensitive fluorescent indicators and UV-triggered fluorescent markers of protein diffusion and trafficking in living cells.

Figure 1

Ribbon diagram of the WT GFP structure. The α-helices are shown in red, the β-strands are shown in green, and the chromophore is shown as a ball-and-stick model. The figure was produced by molscript (29) and raster3d (30, 31).

Figure 2

Schematic diagram of the interactions between the chromophore and the surrounding residues and water molecules in (a) WT GFP and (b) S65T mutant. Hydrogen bonds are shown as dashed lines and have the indicated lengths in Å. The cut-off for the hydrogen bond distance is set to 3.2 Å. (c) Stereoview of the final 2F_o-F_c electron density map of the WT GFP contoured at 1.0 σ, showing chromophore and the surrounding residues. (d) Stereoview of the Thr-203 side chain with the omit electron density (2F_o-F_c) of the WT GFP contoured at 1.0 σ. The side chain was modeled as A (major) and B (minor) conformations with relative occupancies 0.85:0.15.

Figure 2

Schematic diagram of the interactions between the chromophore and the surrounding residues and water molecules in (a) WT GFP and (b) S65T mutant. Hydrogen bonds are shown as dashed lines and have the indicated lengths in Å. The cut-off for the hydrogen bond distance is set to 3.2 Å. (c) Stereoview of the final 2F_o-F_c electron density map of the WT GFP contoured at 1.0 σ, showing chromophore and the surrounding residues. (d) Stereoview of the Thr-203 side chain with the omit electron density (2F_o-F_c) of the WT GFP contoured at 1.0 σ. The side chain was modeled as A (major) and B (minor) conformations with relative occupancies 0.85:0.15.

Figure 3

Proposed mechanism for the photoisomerization of WT GFP based on the structural data and the spectroscopic work (33). State A is the predominant form seen in the WT structure, state B is the form seen in the S65T structure, and state I is the intermediate not seen in either of the two structures. Changes from A to I are indicated in blue, and differences between I and B are in red. The side chains of Arg-96 and Gln-94, which make hydrogen bonds with the carbonyl of the imidazolinone ring, do not show major conformational changes and are not drawn in this figure.

Figure 4

View of the orientation of the hydrogen bonds in (a) the minor form of the WT GFP structure (state B) and (b) the S65T structure. The chromophore and the side chains of Glu-222 and Val-61 are shown as a ball-and-stick model. The nitrogens are shown in blue, oxygens in red, carbons in dark gray, and hydrogens in light gray. The figure was produced by molscript (29).