title: Femtosecond fluorescence spectra of tryptophan in human gamma-crystallin mutants: site-dependent ultrafast quenching.
abstract: The eye lens Crystallin proteins are subject to UV irradiation throughout life, and the photochemistry of damage proceeds through the excited state; thus, their tryptophan (Trp) fluorescence lifetimes are physiologically important properties. The time-resolved fluorescence spectra of single Trps in human gammaD- and gammaS-Crystallins have been measured with both an upconversion spectrophotofluorometer on the 300 fs to 100 ps time scale, and a time correlated single photon counting apparatus on the 100 ps to 10 ns time scale, respectively. Three Trps in each wild type protein were replaced by phenylalanine, leading to single-Trp mutants: W68-only and W156-only of HgammaD- and W72-only and W162-only of HgammaS-Crystallin. These proteins exhibit similar ultrafast signatures: positive definite decay associated spectra (DAS) for 50-65 ps decay constants that indicate dominance of fast, heterogeneous quenching. The quenched population (judged by amplitude) of this DAS differs among mutants. Trps 68, 156 in human gammaD- and Trp72 in human gammaS-Crystallin are buried, but water can reach amide oxygen and ring HE1 atoms through narrow channels. QM-MM simulations of quenching by electron transfer predict heterogeneous decay times from 50-500 ps that agree with our experimental results. Further analysis of apparent radiative lifetimes allow us to deduce that substantial subpopulations of Trp are fully quenched in even faster (sub-300 fs) processes for several of the mutants. The quenching of Trp fluorescence of human gammaD- and gammaS-Crystallin may protect them from ambient light induced photo damage.
authors.affiliation: Optical Spectroscopy Section, Laboratory of Molecular Biophysics, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-1412, USA.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Xu
authors.forename: Jiejin
authors.initials: J
authors.lastname: Chen
authors.forename: Dmitri
authors.initials: D
authors.lastname: Toptygin
authors.forename: Olga
authors.initials: O
authors.lastname: Tcherkasskaya
authors.forename: Patrik
authors.initials: P
authors.lastname: Callis
authors.forename: Jonathan
authors.initials: J
authors.lastname: King
authors.forename: Ludwig
authors.initials: L
authors.lastname: Brand
authors.forename: Jay R
authors.initials: JR
authors.lastname: Knutson
mesh_terms: D006801:Humans; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics; D014466:Ultraviolet Rays; D038222:gamma-Crystallins / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects*
chemical_list: D038222:gamma-Crystallins; D010649:Phenylalanine; D014364:Tryptophan