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J. Chen, S. L. Flaugh, P. R. Callis, J. King; Mechanism of Fluorescence Quenching of Tryptophans in Human S-Crystallin. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2039. doi: https://doi.org/.
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Human γS-crystallin (HγS-Crys) is a stable eye lens protein that is expressed post-natally in the outer layers of the eye lens. It must remain soluble and folded throughout the human lifetime. HγS-Crys has two homologous ß-sheet domains, each containing a pair of highly conserved buried tryptophans. These tryptophans display anomalous fluorescence that is quenched in the native state with respect to the denatured state without the existence of the metal ligands or cofactors. We revealed the mechanism of this quenching by combination of experiments with theoretical calculation.
Based on the fluorescence spectra of triple tryptophan to phenylalanine mutants, the quantum yields of the four tryptophans in HγS-Crys have been explicitly studied. The electron transfer rate constant of four tryptophans were calculated by hybrid quantum mechanical-molecular mechanical (QM-MM) methods base on the x-ray structure of the C-terminal domain of HγS-Crys and the NMR structure of murine γS-crystallin.
Trp72 and Trp162 of HγS-Crys are highly quenched, with quantum yields of 0.013 and 0.03, respectively. Trp46 and Trp136 are moderately fluorescent, with quantum yields of 0.066 and 0.25, respectively. There is energy transfer from Trp136 to Trp162 in the C-terminal domain but not in the N-terminal domain. QM-MM simulations with the four different excited tryptophans as electron donors strongly indicate that electron transfer rates to the amide backbone of Trp162 are extremely fast relative to those for Trp136. Quenching of Trp72/162 is due to the efficient electron transfer from tryptophan indole ring to amide backbone.
The quenching mechanism of HγS-Crys is similar as Human γD-crystallin reported previously. Trps 72/162 display abnormally low fluorescence intensity. QM-MM simulations implicate the environmental charged residues and nearby waters of Trp72/162 favorably stabilize the electron transfer events from excited indole ring to the backbone amide. Förster resonance energy transfer from the strongly (Trp136) to weakly (Trp162) emitting Trp in the C-terminal domain serves to further reduce the overall quantum yields. The backbone conformation of tryptophans in γ-crytallins may have evolved to protect the tryptophans of crystallin proteins from UV-induced photodamage.
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