Mechanism of the highly efficient quenching of tryptophan fluorescence in human γD-crystallin
Mechanism of the highly efficient quenching of tryptophan fluorescence in human γD-crystallin
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DOI：
10.1021/bi060988v
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发表时间：
2006-09-26
期刊：
影响因子：
2.9
通讯作者：
King, Jonathan
中科院分区：
文献类型：
--
作者：
Chen, Jiejin;Flaugh, Shannon L.;King, Jonathan
隐埋色氨酸（Trps）的荧光猝灭是蛋白质构象的重要报告分子。人γ D-晶状体蛋白（H γ D-Crys）是一种非常稳定的眼透镜蛋白，在人的一生中必须保持可溶性和折叠。非天然或共价损伤的H γ D-Crys的聚集与流行的眼病成熟型白内障相关。H gamma D-Crys有两个同源的β折叠结构域，每个结构域包含一对高度保守的埋藏的双链。尽管不存在金属配体或辅因子，Trps的整体荧光在天然状态下被淬灭。我们报告的荧光发射光谱和量子产率的许多定点突变体的H γ D-Crys的详细定量测量的结果。从三重色氨酸到苯丙氨酸突变体的荧光，发现同源对Trp 68和Trp 156被极度淬灭，量子产率接近0.01。同源对Trp 42和Trp 130是中等荧光的，量子产率分别为0.13和0.17。为了鉴定淬灭和/或静电干扰残基，用中性或疏水性残基取代Trp 68和Trp 156周围的一组17个候选氨基酸。这些突变体与其自身背景相比均未显示荧光强度的显著变化。混合量子力学-分子力学（QM-MM）模拟与四个不同的激发Trps作为电子供体强烈表明，电子转移速率的酰胺骨架的Trp 68和Trp 156是非常快的Trp 42和Trp 130。这与实验测量的量子产率一致，并且与不存在淬灭侧链一致。有效的电子转移到骨干是可能的Trp 68和Trp 156，因为净有利的位置，几个带电的残基和附近的沃茨的方向，共同稳定电子转移静电。单和双色氨酸苯丙氨酸突变体的荧光发射光谱提供了强有力的证据，能量转移从Trp 42到Trp 68的N-末端结构域和从Trp 130到Trp 156的C-末端结构域。Hgamma D-Crys中的Dahans的骨架构象可能已经部分地进化以使透镜成为非常有效的UV过滤器，而有效的淬灭提供了原位机制以保护晶状体蛋白的Dahans免于光化学降解。
Quenching of the fluorescence of buried tryptophans (Trps) is an important reporter of protein conformation. Human gamma D-crystallin (H gamma D-Crys) is a very stable eye lens protein that must remain soluble and folded throughout the human lifetime. Aggregation of non-native or covalently damaged H gamma D-Crys is associated with the prevalent eye disease mature-onset cataract. H gamma D-Crys has two homologous beta-sheet domains, each containing a pair of highly conserved buried tryptophans. The overall fluorescence of the Trps is quenched in the native state despite the absence of the metal ligands or cofactors. We report the results of detailed quantitative measurements of the fluorescence emission spectra and the quantum yields of numerous site-directed mutants of H gamma D-Crys. From fluorescence of triple Trp to Phe mutants, the homologous pair Trp68 and Trp156 were found to be extremely quenched, with quantum yields close to 0.01. The homologous pair Trp42 and Trp130 were 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 17 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. This is in agreement with the quantum yields measured experimentally and consistent with the absence of a quenching side chain. 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. The backbone conformation of tryptophans in H gamma D-Crys may have evolved in part to enable the lens to become a very effective UV filter, while the efficient quenching provides an in situ mechanism to protect the tryptophans of the crystallins from photochemical degradation.