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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.

SUBMITTER: Chen J

PROVIDER: S-EPMC2674318 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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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.

Chen Jiejin J Callis Patrik R PR King Jonathan J

Biochemistry 20090501 17

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 t ...[more]

PMID: 19358562

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Project description:Human gammaD-crystallin (HgammaD-Crys) is a two-domain, beta-sheet eye lens protein found in the lens nucleus. Its long-term solubility and stability are important to maintain lens transparency throughout life. HgammaD-Crys has four highly conserved buried tryptophans (Trps), with two in each of the homologous beta-sheet domains. In situ, these Trps will be absorbing ambient UV radiation that reaches the lens. The dispersal of the excited-state energy to avoid covalent damage is likely to be physiologically relevant for the lens crystallins. Trp fluorescence is efficiently quenched in native HgammaD-Crys. Previous steady-state fluorescence measurements provide strong evidence for energy transfer from Trp42 to Trp68 in the N-terminal domain and from Trp130 to Trp156 in the C-terminal domain [Chen, J., et al. (2006) Biochemistry 45, 11552-11563]. Hybrid quantum mechanical-molecular mechanical (QM-MM) simulations indicated that the fluorescence of Trp68 and Trp156 is quenched by fast electron transfer to the amide backbone. Here we report additional information obtained using time-resolved fluorescence spectroscopy. In the single-Trp-containing proteins (Trp42-only, Trp68-only, Trp130-only, and Trp156-only), the highly quenched Trp68 and Trp156 have very short lifetimes, tau approximately 0.1 ns, whereas the moderately fluorescent Trp42 and Trp130 have longer lifetimes, tau approximately 3 ns. In the presence of the energy acceptor (Trp68 or Trp156), the lifetime of the energy donor (Trp42 or Trp130) decreased from approximately 3 to approximately 1 ns. The intradomain energy transfer efficiency is 56% in the N-terminal domain and is 71% in the C-terminal domain. The experimental values of energy transfer efficiency are in good agreement with those calculated theoretically. The absence of a time-dependent red shift in the time-resolved emission spectra of Trp130 proves that its local environment is very rigid. Time-resolved fluorescence anisotropy measurements with the single-Trp-containing proteins, Trp42-only and Trp130-only, indicate that the protein rotates as a rigid body and no segmental motion is detected. A combination of energy transfer with electron transfer results in short excited-state lifetimes of all Trps, which, together with the high rigidity of the protein matrix around Trps, could protect HgammaD-Crys from excited-state reactions causing permanent covalent damage.

Project description:Interaction among crystallins is required for the maintenance of lens transparency. Deamidation is one of the most common post-translational modifications in crystallins, which results in incorrect interaction and leads to aggregate formation. Various studies have established interaction among the ?- and ?-crystallins. Here, we investigated the effects of the deamidation of ?A- and ?B-crystallins on their interaction with ?A3-crystallin using surface plasmon resonance (SPR) and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) methods. SPR analysis confirmed adherence of WT ?A- and WT ?B-crystallins and their deamidated mutants with ?A3-crystallin. The deamidated mutants of ?A-crystallin (?A N101D and ?A N123D) displayed lower adherence propensity for ?A3-crystallin relative to the binding affinity shown by WT ?A-crystallin. Among ?B-crystallin mutants, ?B N78D displayed higher adherence propensity whereas ?B N146D mutant showed slightly lower binding affinity for ?A3-crystallin relative to that shown by WT ?B-crystallin. Under the in vivo condition (FLIM-FRET), both ?A-deamidated mutants (?A N101D and ?A N123D) exhibited strong interaction with ?A3-crystallin (32±4% and 36±4% FRET efficiencies, respectively) compared to WT ?A-crystallin (18±4%). Similarly, the ?B N78D and ?B N146D mutants showed strong interaction (36±4% and 22±4% FRET efficiencies, respectively) with ?A3-crystallin compared to 18±4% FRET efficiency of WT ?B-crystallin. Further, FLIM-FRET analysis of the C-terminal domain (CTE), N-terminal domain (NTD), and core domain (CD) of ?A- and ?B-crystallins with ?A3-crystallin suggested that interaction sites most likely reside in the ?A CTE and ?B NTD regions, respectively, as these domains showed the highest FRET efficiencies. Overall, results suggest that similar to WT ?A- and WT?B-crystallins, the deamidated mutants showed strong interactionfor ?A3-crystallin. Variable in vitro and in vivo interactions are most likely due to the mutant's large size oligomers, reduced hydrophobicity, and altered structures. Together, the results suggest that deamidation of ?-crystallin may facilitate greater interaction and the formation of large oligomers with other crystallins, and this may contribute to the cataractogenic mechanism.

Dataset Information

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.

Publications

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.

Similar Datasets

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