| Abstract
| - Quenching of the fluorescence of buried tryptophans (Trps) is an important reporter of proteinconformation. Human γD-crystallin (HγD-Crys) is a very stable eye lens protein that must remain solubleand folded throughout the human lifetime. Aggregation of non-native or covalently damaged HγD-Crysis associated with the prevalent eye disease mature-onset cataract. HγD-Crys has two homologous β-sheetdomains, each containing a pair of highly conserved buried tryptophans. The overall fluorescence of theTrps is quenched in the native state despite the absence of the metal ligands or cofactors. We report theresults of detailed quantitative measurements of the fluorescence emission spectra and the quantum yieldsof numerous site-directed mutants of HγD-Crys. From fluorescence of triple Trp to Phe mutants, thehomologous pair Trp68 and Trp156 were found to be extremely quenched, with quantum yields close to0.01. The homologous pair Trp42 and Trp130 were moderately fluorescent, with quantum yields of 0.13and 0.17, respectively. In an attempt to identify quenching and/or electrostatically perturbing residues, aset of 17 candidate amino acids around Trp68 and Trp156 were substituted with neutral or hydrophobicresidues. None of these mutants showed significant changes in the fluorescence intensity compared totheir own background. Hybrid quantum mechanical−molecular mechanical (QM-MM) simulations withthe four different excited Trps as electron donors strongly indicate that electron transfer rates to the amidebackbone of Trp68 and Trp156 are extremely fast relative to those for Trp42 and Trp130. This is inagreement with the quantum yields measured experimentally and consistent with the absence of a quenchingside chain. Efficient electron transfer to the backbone is possible for Trp68 and Trp156 because of the netfavorable location of several charged residues and the orientation of nearby waters, which collectivelystabilize electron transfer electrostatically. The fluorescence emission spectra of single and double Trp toPhe mutants provide strong evidence for energy transfer from Trp42 to Trp68 in the N-terminal domainand from Trp130 to Trp156 in the C-terminal domain. The backbone conformation of tryptophans inHγD-Crys may have evolved in part to enable the lens to become a very effective UV filter, while theefficient quenching provides an in situ mechanism to protect the tryptophans of the crystallins fromphotochemical degradation.
|
