首页> 美国卫生研究院文献>other >The Key to the Extraordinary Thermal Stability of P. furiosus Holo-Rubredoxin: Iron Binding-Guided Packing of a Core Aromatic Cluster Responsible for High Kinetic Stability of the Native Structure
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The Key to the Extraordinary Thermal Stability of P. furiosus Holo-Rubredoxin: Iron Binding-Guided Packing of a Core Aromatic Cluster Responsible for High Kinetic Stability of the Native Structure

机译:P. furiosus Holo-Rubredoxin的非凡热稳定性的关键:负责天然结构的高动力学稳定性的核心芳香簇的铁结合引导填料

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摘要

Pyrococcus furiosus rubredoxin (PfRd), a small, monomeric, 53 residues-long, iron-containing, electron-transfer protein of known structure is sometimes referred to as being the most structurally-stable protein known to man. Here, using a combination of mutational and spectroscopic (CD, fluorescence, and NMR) studies of differently made holo- and apo-forms of PfRd, we demonstrate that it is not the presence of iron, or even the folding of the PfRd chain into a compact well-folded structure that causes holo-PfRd to display its extraordinary thermal stability, but rather the correct iron binding-guided packing of certain residues (specifically, Trp3, Phe29, Trp36, and also Tyr10) within a tight aromatic cluster of six residues in PfRd's hydrophobic core. Binding of the iron atom appears to play a remarkable role in determining subtle details of residue packing, forcing the chain to form a hyper-thermally stable native structure which is kinetically stable enough to survive (subsequent) removal of iron. On the other hand, failure to bind iron causes the same chain to adopt an equally well-folded native-like structure which, however, has a differently-packed aromatic cluster in its core, causing it to be only as stable as any other ordinary mesophile-derived rubredoxin. Our studies demonstrate, perhaps for the very first time ever that hyperthermal stability in proteins can owe to subtle differences in residue packing vis a vis mesostable proteins, without there being any underlying differences in either amino acid sequence, or bound ligand status.
机译:激烈热球菌氧化还原酶(PfRd)是一种已知结构的小单体长53个残基,含铁的电子转移蛋白,有时被称为人类已知的结构最稳定的蛋白。在这里,结合使用突变和分光镜(CD,荧光和NMR)研究不同形式的PfRd的完整形式和脱辅基形式,我们证明它不存在铁,甚至不存在PfRd链折叠成紧密折叠的紧凑结构,使holo-PfRd表现出非凡的热稳定性,而是在紧密的芳族簇(六簇)中正确引导铁结合,以某些残基(特别是Trp3,Phe29,Trp36和Tyr10)正确包裹PfRd疏水核心中的残基。铁原子的结合似乎在确定残基堆积的细微细节方面起着显著作用,迫使该链形成超热稳定的天然结构,该结构在动力学上足以保持(随后)去除铁。另一方面,未能结合铁会导致同一链采用同样折叠的天然样结构,但是其核心具有不同堆积的芳族簇,从而使其仅与其他任何普通分子一样稳定嗜温菌来源的氧化还原酶。我们的研究表明,蛋白质的高温稳定性可能是有史以来第一次,这归因于残基相对于可降解蛋白质的堆积存在细微差异,而氨基酸序列或结合配体状态没有任何根本差异。

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