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首页> 外文期刊>Biochemistry >Unfolding and conformational studies on bovine adrenodoxin probed by engineered intrinsic tryptophan fluorescence.
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Unfolding and conformational studies on bovine adrenodoxin probed by engineered intrinsic tryptophan fluorescence.

机译：工程化的固有色氨酸荧光探针探测牛肾上腺毒素的展开和构象研究。

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摘要

An intrinsic steady-state fluorescent system for bovine adrenodoxin has been developed to study the protein structure in solution and the processes involved in protein unfolding. Since mature Adx contains no natural Trp residue as internal probe, all of the aromatic amino acids, tyrosine at position 82 and four phenylalanines at positions 11, 43, 59 and 64, were at each case replaced by tryptophan. The resulting single tryptophan containing mutants kept their biological function compared with the wild type. Molecular modeling studies verify thermal unfolding experiments which point to a dramatically reduced stability caused by steric hindrance only for mutant F59W. Fluorescence spectra, Stern-Volmer quenching constants, and fluorescence energy transfer calculations indicated the analyzed positions to be situated in solution in the same immediate environment as in the crystal structure. Unfolding experiments with Gdn-HCl and time-resolved stopped-flow measurements provide evidence for differential stability and a chronologically ordered unfolding mechanism of the different fluorescence probe positions in the protein.
机译：已开发出用于牛肾上腺毒素的内在稳态荧光系统，以研究溶液中的蛋白质结构以及涉及蛋白质展开的过程。由于成熟的Adx不包含天然Trp残基作为内部探针，因此在每种情况下，所有的芳香族氨基酸，82位的酪氨酸和11、43、59和64位的四个苯丙氨酸都被色氨酸取代。与野生型相比，所得的含有单个色氨酸的突变体保持了其生物学功能。分子模型研究验证了热展开实验，该实验表明仅因突变体F59W而由位阻引起的稳定性大大降低。荧光光谱，Stern-Volmer猝灭常数和荧光能量转移计算表明，所分析的位置位于溶液中与晶体结构相同的直接环境中。用Gdn-HCl进行的展开实验和时间分辨的停流测量为蛋白质中不同荧光探针位置的差异稳定性和按时间顺序排列的展开机制提供了证据。

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