Local Conformational Constraint of Firefly Luciferase Can Affect the Energy of Bioluminescence and Enzyme Stability.
Local Conformational Constraint of Firefly Luciferase Can Affect the Energy of Bioluminescence and Enzyme Stability.
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DOI：
10.31635/ccschem.022.202101733
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发表时间：
2022-05
期刊：
影响因子：
11.2
通讯作者：
Hecht, Sidney M.
中科院分区：
文献类型：
--
作者：
Zhang, Chao;Bai, Xiaoguang;Chen, Shengxi;Dedkova, Larisa M.;Hecht, Sidney M.
关键词：
构象动力学对酶催化有重要的贡献，因此有针对性的构象约束可能会影响催化。萤火虫荧光素酶在催化过程中发生广泛的结构变化；关键残留物形成疏水性口袋，排除水并最大限度地产生能量光。点突变体几乎总是比野生型发出更长的波长（更低的能量）。构象约束，在优化激发态结构的关键位置使用二肽类似物3，产生荧光素酶在约10 nm的短波长发射。相比之下，引入构象约束的类似物4,5或7使荧光素酶在更长的波长下发射，而相关的无约束荧光素酶（类似物6）则表现出野生型发射。约束型荧光素酶比野生型更稳定。蛋白质模型表明，构象受限二肽的“内”或“外”取向分别导致较短或较长的发射波长。更广泛地说，这些结果表明，在体外和体内，局部构象约束可以控制酶行为的特定元素。这代表了通过在特定蛋白质位置引入构象约束二肽来研究酶功能的第一个例子。本文所发现的荧光素酶修饰的原理将使研究能够控制修饰的荧光素酶的波长发射和光物理性质。
Conformational dynamics contribute importantly to enzyme catalysis, such that targeted conformational constraint may affect catalysis. Firefly luciferases undergo extensive structural change during catalysis; key residues form a hydrophobic pocket, excluding water and enabling maximally energetic light production. Point mutants almost always luminesce at longer wavelengths (lower energy) than the wild type. Conformational constraint, using dipeptide analogue 3 at a position critical for optimized excited state structure, produced luciferase emission at a shorter wavelength by ~10 nm. In comparison, introduction of conformationally constrained analogues 4, 5, or 7 afforded luciferases emitting at longer wavelengths, while a related unconstrained luciferase (analogue 6) exhibited wild-type emission. The constrained luciferases tested were more stable than the wild type. Protein modeling demonstrated that the “inside” or “outside” orientation of the conformationally constrained dipeptide led to the shorter or longer emission wavelength, respectively. More broadly, these results suggest that local conformational constraint can control specific elements of enzyme behavior, both in vitro and in vivo. This represents the first example of studying enzyme function by introducing conformationally constrained dipeptides at a specific protein position. The principles discovered here in luciferase modification will enable studies to control the wavelength emission and photophysical properties of modified luciferases.
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影响因子：
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作者：
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通讯作者：
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