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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Demonstration of a low-energy on-pathway intermediate in a fast-folding protein by kinetics, protein engineering, and simulation
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Demonstration of a low-energy on-pathway intermediate in a fast-folding protein by kinetics, protein engineering, and simulation

机译：通过动力学，蛋白质工程和模拟论证快速折叠蛋白质中的低能量通路中间体

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摘要

It is controversial whether fast-folding proteins can form productive on-pathway intermediates that are more stable than the denatured state because noncovalent intermediates are usually evanescent. Here, we apply the classical criteria for the existence of intermediates: namely, the intermediates form and react rapidly enough to be on pathway and they can be isolated and characterized. The folding of the 71-residue, mainly alpha-helical FF domain from human HYPA/FBP11 fulfills these classical criteria, as was found for Im7. The FF domain folds in two phases, one on the mus and the other on the ms time scale. An engineered mutant folds only to a partly folded state, with some 20-40% of the native helical content. The kinetic properties of the mutant are identical to those found for the fast phase of the wild-type protein, and it is likely that the mutant folds just to the intermediate state. A full kinetic analysis of the folding of wild-type protein, using the amplitudes of its native and denatured states and the observed values for the mutant, rules out an off-pathway scheme but fits an on-pathway scheme, with a low energy intermediate that is modeled by the mutant. The experimental proof benchmarks a molecular dynamics method that identifies an obligatory intermediate observed in multiple simulations. The conformational space defining this intermediate is visited several times in the simulations, leading to high populations consistent with the presence of a low energy intermediate.
机译：由于非共价中间体通常是渐逝性的，因此快速折叠的蛋白质是否可以形成比变性状态更稳定的生产性中间中间体仍存在争议。在这里，我们对中间体的存在应用经典标准：即，中间体形成并反应迅速，足以进入途径，并且可以对其进行分离和表征。如从Im7中发现的，来自人HYPA / FBP11的71个残基（主要是α-螺旋FF域）的折叠符合这些经典标准。 FF域分两个阶段折叠，一个阶段在mus阶段，另一个阶段在ms时间尺度上。工程突变体仅折叠至部分折叠状态，天然螺旋含量约为20％至40％。突变体的动力学特性与野生型蛋白质快相的动力学特性相同，并且可能突变体仅折叠到中间状态。使用其天然和变性状态的幅度以及突变体的观察值，对野生型蛋白质折叠进行完整的动力学分析，排除了偏路径方案，但适合沿路径方案，且中间能量低由突变体建模。实验证明以分子动力学方法为基准，该方法确定了在多次模拟中观察到的强制性中间产物。在模拟中，多次访问了定义该中间体的构象空间，从而导致高种群与低能中间体的存在相一致。

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