Mechanism of the Efficient Tryptophan Fluorescence Quenching in Human γD-Crystallin Studied by Time-Resolved Fluorescence

Abstract

Human γD-crystallin (HγD-Crys) is a two-domain, β-sheet eye lens protein found in the lens nucleus. Its long-term solubility and stability are important to maintain lens transparency throughout life. HγD-Crys has four highly conserved buried tryptophans (Trps), with two in each of the homologous β-sheet domains. In situ, these Trps will be absorbing ambient UV radiation that reaches the lens. The dispersal of the excited-state energy to avoid covalent damage is likely to be physiologically relevant for the lens crystallins. Trp fluorescence is efficiently quenched in native HγD-Crys. Previous steady-state fluorescence measurements 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 [Chen, J., et al. (2006) Biochemistry 45, 11552−11563]. Hybrid quantum mechanical−molecular mechanical (QM-MM) simulations indicated that the fluorescence of Trp68 and Trp156 is quenched by fast electron transfer to the amide backbone. Here we report additional information obtained using time-resolved fluorescence spectroscopy. In the single-Trp-containing proteins (Trp42-only, Trp68-only, Trp130-only, and Trp156-only), the highly quenched Trp68 and Trp156 have very short lifetimes, τ ∼0.1 ns, whereas the moderately fluorescent Trp42 and Trp130 have longer lifetimes, τ ∼3 ns. In the presence of the energy acceptor (Trp68 or Trp156), the lifetime of the energy donor (Trp42 or Trp130) decreased from ∼3 to ∼1 ns. The intradomain energy transfer efficiency is 56% in the N-terminal domain and is 71% in the C-terminal domain. The experimental values of energy transfer efficiency are in good agreement with those calculated theoretically. The absence of a time-dependent red shift in the time-resolved emission spectra of Trp130 proves that its local environment is very rigid. Time-resolved fluorescence anisotropy measurements with the single-Trp-containing proteins, Trp42-only and Trp130-only, indicate that the protein rotates as a rigid body and no segmental motion is detected. A combination of energy transfer with electron transfer results in short excited-state lifetimes of all Trps, which, together with the high rigidity of the protein matrix around Trps, could protect HγD-Crys from excited-state reactions causing permanent covalent damage.