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首页> 外文OA文献 >Probing folding and fluorescence quenching in human γD crystallin Greek key domains using triple tryptophan mutant proteins
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Probing folding and fluorescence quenching in human γD crystallin Greek key domains using triple tryptophan mutant proteins

机译：使用三色氨酸突变蛋白探测人γD晶状体希腊语关键域中的折叠和荧光猝灭

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摘要

Human γD crystallin (HγD-Crys), a major component of the human eye lens, is a 173-residue, primarily β-sheet protein, associated with juvenile and mature-onset cataracts. HγD-Crys has four tryptophans, with two in each of the homologous Greek key domains, which are conserved throughout the γ-crystallin family. HγD-Crys exhibits native-state fluorescence quenching, despite the absence of ligands or cofactors. The tryptophan absorption and fluorescence quenching may influence the lens response to ultraviolet light or the protection of the retina from ambient ultraviolet damage. To provide fluorescence reporters for each quadrant of the protein, triple mutants, each containing three tryptophan-to-phenylalanine substitutions and one native tryptophan, have been constructed and expressed. Trp 42-only and Trp 130-only exhibited fluorescence quenching between the native and denatured states typical of globular proteins, whereas Trp 68-only and Trp 156-only retained the anomalous quenching pattern of wild-type HγD-Crys. The three-dimensional structure of HγD-Crys shows Tyr/Tyr/His aromatic cages surrounding Trp 68 and Trp 156 that may be the source of the native-state quenching. During equilibrium refolding/unfolding at 37°C, the tryptophan fluorescence signals indicated that domain I (W42-only and W68-only) unfolded at lower concentrations of GdnHCl than domain II (W130-only and W156-only). Kinetic analysis of both the unfolding and refolding of the triple-mutant tryptophan proteins identified an intermediate along the HγD-Crys folding pathway with domain I unfolded and domain II intact. This species is a candidate for the partially folded intermediate in the in vitro aggregation pathway of HγD-Crys.
机译：人γD晶状体蛋白（HγD-Crys）是人眼晶状体的主要成分，是一种173残基的蛋白质，主要是β-折叠蛋白，与青少年和成熟期白内障相关。 HγD-Crys具有四个色氨酸，每个同源希腊语关键域中有两个，在整个γ-晶状蛋白家族中都保守。尽管没有配体或辅因子，HγD-Crys仍表现出天然状态的荧光猝灭。色氨酸的吸收和荧光猝灭可能会影响晶状体对紫外线的反应或保护视网膜免受周围紫外线的损害。为提供蛋白质每个象限的荧光报告基因，已构建并表达了三个突变体，每个突变体均包含三个色氨酸至苯丙氨酸取代基和一个天然色氨酸。仅Trp 42和仅Trp 130在典型的球形蛋白质的天然状态和变性状态之间表现出荧光猝灭，而仅Trp 68和仅Trp 156保留了野生型HγD-Crys的异常猝灭模式。 HγD-Crys的三维结构显示Tyr / Tyr / His芳香族笼子围绕着Trp 68和Trp 156，它们可能是自然态猝灭的来源。在37°C的平衡重折叠/展开过程中，色氨酸荧光信号表明结构域I（仅W42和W68）在GdnHCl浓度低于结构域II（仅W130和W156）时展开。对三突变色氨酸蛋白的展开和再折叠进行动力学分析，确定了沿着HγD-Crys折叠途径的中间体，其中I区未折叠，II区完整。该物种是HγD-Crys体外聚集途径中部分折叠的中间体的候选者。

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