BIOL 42 |
| Human γD-crystallin (HγD-Crys) is a very stable eye lens protein that must remain soluble and folded throughout the human lifetime. HγD-Crys has two homologous β-sheet domains, each containing a pair of highly conserved buried tryptophans (Trps). The overall fluorescence of Trps is quenched in the native state. We report the results of detailed quantitative measurements of the fluorescence emission spectra and the quantum yields of 51 site-directed mutants of HγD-Crys. From triple Trps to Phenylalanine mutants, Trp68 and Trp156 are found to be extremely quenched, with quantum yields close to 0.01. Trp42 and Trp130 are moderately fluorescent, with quantum yields of 0.13 and 0.17, respectively. In an attempt to identify quenching and/or electrostatically perturbing residues, a set of candidate amino acids around Trp68 and Trp156 were substituted with neutral or hydrophobic residues. None of these mutants showed significant changes in the fluorescence intensity compared to their own background. Hybrid quantum mechanical-molecular mechanical (QM-MM) simulations with the four different excited Trps as electron donors strongly indicate that electron transfer rates to the amide backbone of Trp68 and Trp156 are extremely fast relative to those for Trp42 and Trp130, in agreement with the quantum yields measured experimentally and consistent with the absence of a quenching sidechain. Efficient electron transfer to the backbone is possible for Trp68 and Trp156 because of the net favorable location of several charged residues and the orientation of nearby waters, which collectively stabilize electron transfer electrostatically. The fluorescence emission spectra of single and double Trp to Phe mutants 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. Quenching may be an in situ mechanism to protect the Trps of lens crystallin proteins from photochemical degradation. |
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Protein Structure and Folding
4:30 PM-6:30 PM, Sunday, 10 September 2006 Moscone Center -- Hall D, Poster
Division of Biological Chemistry |