Proton pathways in green fluorescence protein

Abstract

Proton pathways in green fluorescent protein (GFP) are more extended than previously reported. In the x-ray data of wild-type GFP, a two-step exit pathway exists from the active site to the protein surface, controlled by a threonine switch. A proton entry pathway begins at a glutamate-lysine cluster around Glu-5, and extends all the way to the buried Glu-222 near the active site. This structural evidence suggests that GFP may function as a portable light-driven proton-pump, with proton emitted in the excited state through the switchable exit pathway, and replenished from Glu-222 and the Glu-5 entry pathway in the ground state.

FIGURE 1

Time-resolved fluorescence from wt-GFP (293 K, in pH = 8 buffer). Circles are experimental data by Leiderman et al. (2004), showing the time dependence of the 450 nm emission intensity, I, corrected for the fluorescence lifetime, τ_f = 2.4 ns (determined from the decay of the anion emission at 510 nm). These data are from a different experimental run than shown in their Fig. 5, and reproduced here with permission. Dashed and dash-dot lines represent the asymptotic t⁻¹ and t^−3/2 laws, respectively, convoluted with the instrument response function (dotted). Both powers-laws were multiplied by the same arbitrary prefactor. Note the log-log scale.

FIGURE 2

X-ray structure of the anionic (B-state) GFP reveals a hydrogen-bond escape pathway for the proton, leading from the chromophore (Tyr-66) to the OH of Thr-203 and then to the bb-carbonyl of His-148, which is already on the surface of the protein. The HB lengths (O⋯O distance) are given in angstroms (for comparison, the typical distance for liquid water is 2.85 Å). Color codes: oxygen, red; nitrogen, blue; carbon, gray; and added hydrogens, white; bb connections, black. Coordinates from PDB file 1Q4A; x-ray diffraction measurements of Jain and Ranganathan (2004).

FIGURE 3

Proton escape hole on the surface of B-state GFP, after removing a few surface water molecules for better visualization. While the Cro is in its excited state, the proton can hop along the pathway denoted 1, 2, 3, corresponding to the three relevant oxygen atoms of Tyr-66 (black), Thr-203, and His-148. Also seen are Glu-222 (red, under Phe-223 and Gln-204), Ser-205, “the” water molecule (w, white), and the 202–204 triplet (see text). The space filling view is color coded by amino-acid side-chain characteristics: Greens, aliphatic; gray, aromatic; red, carboxylic (Glu, Asp); dark blues, charged amino (Arg, Lys, His); cyan, uncharged amino; yellow, sulfur; and orange, Ser and Thr. Water oxygens are gray. Coordinates from PDB file 1Q4A.

FIGURE 4

A more extended exit channel may continue from the bb-carbonyl of His-148 along various solvent-protruding side chains or surface water molecules. Interatom distances in angstroms. Color codes as in Fig. 2. Coordinates from PDB file 1Q4A.

FIGURE 5

Formation of a transient proton escape pathway in wt-GFP. Panel A depicts the acidic state A of a wt-GFP from PDB file 1GFL (Yang et al., 1996). Here the Thr-203 switch is “off” and no exit pathway exists. See text for detailed discussion of the HB network. O⋯O distances in angstroms and O–H⋯O angles in degrees. The three shown internal rotations lead to state B′, resembling state B of the S65T mutant in Fig. 2. In particular, χ₁ of Thr-203 was varied from −169° to −50° and for His-148 from −53° to −40°. This establishes a transient proton escape route (blue). Color codes as in Fig. 2, with bb continuation in black. Only Thr-203 and His-148 are rendered with all their atoms (including added hydrogens, not seen in the x-ray structure), the other three amino-acids are represented by selected atoms.

SCHEME 1

The two-state threonine switch.

FIGURE 6

A detailed view of the water molecule environment in the anionic B-state (PDB file 1Q4A). HBs (with their orientation) are depicted by blue arrows. Two additional distances are also given.

FIGURE 7

A well-defined proton entry pathway exists in GFP, and it shows a remarkable resemblance to the D-pathway in CcO. The suggested pathway is depicted by dashed arrows, with distances in angstroms, as measured using the coordinates from PDB file 1GFL. No hydrogens are depicted here. The problematic steps involving W255 are denoted in red. Curiously enough, only the part of the HB-net between W60 and Tyr-66 was depicted in previous publications.

FIGURE 8

A bb-nitrogen wire could serve as an alternative to W255 in bridging the gap between Ser-72 and W60. The nitrogens also couple to the bb-carbonyls of Val-68 and Gln-69, allowing several shortcuts to be taken. Coordinates from PDB file 1GFL.

FIGURE 9

Entry point to the proton pathway of Fig. 7 near the N-terminal on the GFP surface. Coordinates from PDB file 1EMB (Brejc et al., 1997) for monomeric GFP were used, because dimerization in file 1GFL occurs close to the depicted protein surface. The space filling view is color coded as in Fig. 3.

FIGURE 10

An additional HB pathway for proton entry is revealed in the S65T, B-state structure (file 1Q4A).

FIGURE 11

A glutamate ring observed on the back surface of the GFP may be functioning as a proton collecting antenna for the Glu-115 pathway in the S65T mutant (file 1Q4A).