The denatured state of several proteins has been shown to display transient structures that are relevant for folding, stability, and aggregation. To detect them by nuclear magnetic resonance (NMR) spectroscopy, the denatured state must be stabilized by chemical agents or changes in temperature. This makes the environment different from that experienced in biologically relevant processes. Using high-resolution heteronuclear NMR spectroscopy, we have characterized several denatured states of a monomeric variant of HIV-1 protease, which is natively structured in water, induced by different concentrations of urea, guanidinium chloride, and acetic acid. We have extrapolated the chemical shifts and the relaxation parameters to the denaturant-free denatured state at native conditions, showing that they converge to the same values. Subsequently, we characterized the conformational properties of this biologically relevant denatured state under native conditions by advanced molecular dynamics simulations and validated the results by comparison to experimental data. We show that the denatured state of HIV-1 protease under native conditions displays rich patterns of transient native and non-native structures, which could be of relevance to its guidance through a complex folding process.

中文翻译：

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天然条件下 HIV-1 蛋白酶的变性状态

几种蛋白质的变性状态已显示出与折叠、稳定性和聚集相关的瞬态结构。为了通过核磁共振 (NMR) 光谱检测它们，变性状态必须通过化学试剂或温度变化来稳定。这使得环境不同于生物相关过程中所经历的环境。使用高分辨率异核 NMR 光谱，我们已经表征了 HIV-1 蛋白酶的单体变体的几种变性状态，该变体在水中天然结构化，由不同浓度的尿素、氯化胍和乙酸诱导。我们已经将化学位移和弛豫参数外推到天然条件下无变性剂的变性状态，表明它们收敛到相同的值。随后，我们通过先进的分子动力学模拟在天然条件下表征了这种生物学相关变性状态的构象特性，并通过与实验数据的比较验证了结果。我们表明，HIV-1 蛋白酶在天然条件下的变性状态显示出丰富的瞬时天然和非天然结构模式，这可能与其通过复杂折叠过程的指导有关。