Mechanism of the very efficient quenching of tryptophan fluorescence in human gamma D- and gamma S-crystallins: the gamma-crystallin fold may have evolved to protect tryptophan residues from ultraviolet photodamage

Abstract

Proteins exposed to UV radiation are subject to irreversible photodamage through covalent modification of tryptophans (Trps) and other UV-absorbing amino acids. Crystallins, the major protein components of the vertebrate eye lens that maintain lens transparency, are exposed to ambient UV radiation throughout life. The duplicated beta-sheet Greek key domains of beta- and gamma-crystallins in humans and all other vertebrates each have two conserved buried Trps. Experiments and computation showed that the fluorescence of these Trps in human gammaD-crystallin is very efficiently quenched in the native state by electrostatically enabled electron transfer to a backbone amide [Chen et al. (2006) Biochemistry 45, 11552-11563]. This dispersal of the excited state energy would be expected to minimize protein damage from covalent scission of the excited Trp ring. We report here both experiments and computation showing that the same fast electron transfer mechanism is operating in a different crystallin, human gammaS-crystallin. Examination of solved structures of other crystallins reveals that the Trp conformation, as well as favorably oriented bound waters, and the proximity of the backbone carbonyl oxygen of the n - 3 residues before the quenched Trps (residue n), are conserved in most crystallins. These results indicate that fast charge transfer quenching is an evolved property of this protein fold, probably protecting it from UV-induced photodamage. This UV resistance may have contributed to the selection of the Greek key fold as the major lens protein in all vertebrates.

Figure 1

Homologous positions of four Trps in γ-crystallins. (A) The crystal structure of human γD-crystallin is shown as a ribbon diagram with four Trps in space-fill: Trp42 and Trp68 in the N-terminal domain and Trp130 and Trp156 in the C-terminal domain (PDB code 1HK0). (B) The NMR structure of murine γS-crystallin is depicted in ribbon representation showing the four Trps in space-fill: Trp46 and Trp72 in the N-terminal domain and Trp136 and Trp162 in the C-terminal domain (PDB code 1ZWM).

Figure 2

Fluorescence emission spectra of native and denatured wild type and various Trp mutants of HγS-Crys. (A) Fluorescence emission spectra of native Trp46-only (◼), Trp72-only (●), and Trp46/Trp72 (▼) and denatured Trp46-only (◻), Trp72-only (○), and Trp46/Trp72 (▽). (B) Fluorescence emission spectra of native Trp136-only (◼), Trp162-only (●), and Trp136/Trp162 (▼) and denatured Trp136-only (◻), Trp162-only (○), and Trp136/Trp162 (▽). (C) Fluorescence emission spectra of native wild type (WT) (◼), W72F (●), and W162F (▼) and denatured wild type (◻), W72F (○), and W162F (▽). (D) Fluorescence emission spectra of native wild type (◼), W46F (●), and W136F (▼) and denatured wild type (◻), W46F (○), and W136F (▽). The solid lines represent the emission spectra of native proteins, and the dotted lines represent the unfolded proteins. Native proteins were incubated in S buffer, and unfolded proteins were incubated in S buffer plus 5.5 M GuHCl for 6 h at 37 °C. The excitation wavelength was at 300 nm, and the protein concentration was 2.75 μM. The buffer signal was subtracted from all spectra. Because the crystal structure of full-length HγS-Crys is not available, the ribbon structure of murine γS-crystallin with Trps in space-fill is shown here instead at the upper right corner.

Figure 3

QM-MM trajectories showing transition energies for the fluorescing state (red) and the CT state (black) for Trps 42 and 68 of HγD-Crys. The relative gap between the states is the main determinant of electron transfer based fluorescence quenching. The energy scale is in kcm⁻¹ (8 kcm⁻¹ = 1 eV = 96.5 kJ/mol). Fluctuations on the gap are on the order of 1 eV.

Figure 4

Representative conserved nearby waters and aromatic residues of quenched Trps in HγD-Crys. (A) The two crystallographic water molecules (yellow oxygens) that most stabilize the electron transfer from the indole ring to the amide backbone of Trp68 in the HγD-Crys (PDB code 1HK0). These sites do not appear to be accessible, but Wat132 is trapped by an additional H-bond to Gln142 and/or Phe56, and Wat137 is usually also H-bonded to Ser72. (B) Representative conserved proximity of the backbone carbonyl bond of n − 3 residues before the nonfluorescent Trps (residue n) in the HγD-Crys.