Femtosecond Fluorescence Spectra of Tryptophan in Human γ-Crystallin Mutants: Site-Dependent Ultrafast Quenching

• Published in: Journal of the American Chemical Society 2009-11, Vol.131 (46), p.16751-16757
• Main Authors: Xu, Jianhua, Chen, Jiejin, Toptygin, Dmitri, Tcherkasskaya, Olga, Callis, Patrik, King, Jonathan, Brand, Ludwig, Knutson, Jay R
• Format: Article
• Language: English
• Subjects: gamma-Crystallins - chemistry; gamma-Crystallins - genetics; gamma-Crystallins - radiation effects; Humans; Phenylalanine - chemistry; Phenylalanine - genetics; Spectrometry, Fluorescence; Tryptophan - chemistry; Tryptophan - genetics; Ultraviolet Rays
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja904857t

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 γD- and γS-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 HγD- and W72-only and W162-only of HγS-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 γD- and Trp72 in human γS-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 γD- and γS-Crystallin may protect them from ambient light induced photo damage.