Mechanism of the efficient tryptophan fluorescence quenching in human gammaD-crystallin studied by time-resolved fluorescence.
Mechanism of the efficient tryptophan fluorescence quenching in human gammaD-crystallin studied by time-resolved fluorescence.
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DOI：
10.1021/bi800499k
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发表时间：
2008-10-07
期刊：
影响因子：
2.9
通讯作者：
King J
中科院分区：
文献类型：
--
作者：
Chen J;Toptygin D;Brand L;King J
人 γD-晶状体蛋白 (HγD-Crys) 是一种存在于晶状体核中的双结构域 β-折叠眼晶状体蛋白。其长期溶解度和稳定性对于在整个生命周期内保持镜片透明度非常重要。 HγD-Crys 具有四个高度保守的隐藏色氨酸 (Trps)，每个同源 β-折叠结构域中有两个。在原位，这些色氨酸将吸收到达镜片的环境紫外线辐射。激发态能量的分散以避免共价损伤可能与晶状体蛋白的生理相关。 Trp 荧光在天然 HγD-Crys 中被有效猝灭。先前的稳态荧光测量为 N 端结构域中从 Trp42 到 Trp68 以及 C 端结构域中从 Trp130 到 Trp156 的能量转移提供了强有力的证据 [Chen, J., et al. 2017]。 (2006) 生物化学 45, 11552−11563]。混合量子力学-分子力学 (QM-MM) 模拟表明，Trp68 和 Trp156 的荧光通过快速电子转移到酰胺主链而被猝灭。在这里，我们报告使用时间分辨荧光光谱获得的附加信息。在含单色氨酸的蛋白质（仅Trp42、仅Trp68、仅Trp130和仅Trp156）中，高度猝灭的Trp68和Trp156具有非常短的寿命，τ∼0.1ns，而中等荧光的Trp42和Trp130具有较长的寿命，τ∼3ns。在能量受体（Trp68 或 Trp156）存在的情况下，能量供体（Trp42 或 Trp130）的寿命从~3 ns减少到~1 ns。 N端域的域内能量转移效率为56%，C端域为71%。能量传输效率的实验值与理论计算值吻合较好。 Trp130 的时间分辨发射光谱不存在与时间相关的红移，这证明其局部环境非常严格。对含单 Trp 的蛋白质（仅 Trp42 和仅 Trp130）进行的时间分辨荧光各向异性测量表明，该蛋白质作为刚体旋转，并且未检测到节段运动。能量转移与电子转移的结合导致所有色氨酸的激发态寿命较短，再加上色氨酸周围蛋白质基质的高刚性，可以保护 HγD-Crys 免受激发态反应造成永久性共价损伤。
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.
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