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首页> 外文期刊>Biophysical Chemistry: An International Journal Devoted to the Physical Chemistry of Biological Phenomena >Kinetic folding studies of the P22 tailspike beta-helix domain reveal multiple unfolded states.
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Kinetic folding studies of the P22 tailspike beta-helix domain reveal multiple unfolded states.

机译：P22尾钉β-螺旋结构域的动力学折叠研究显示了多个未折叠状态。

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摘要

The beta-helix is an important protein fold in many pathogens, and is a challenging system for folding pathway prediction because it primarily is stabilized by non-local interactions along the primary sequence. A useful experimental model of this fold is a monomeric truncation of P22 tailspike protein, the beta-helix domain (bhx). This report describes a systematic in vitro study of the chemical denaturation and refolding of bhx. Results from equilibrium chemical denaturation experiments were consistent with a two-state folding mechanism, but showed only partial reversibility. Stopped-flow fluorescence studies showed a single unfolding step, but two refolding steps. The slow refolding step could be partly attributed to proline isomerization, based on an increased rate during refolding in the presence of PPIase and an increased relative amplitude of this step with increasing delay time in double-jump refolding experiments observed over delays of 5-100 s. However, double-jump refolding experiments with delay times longer than 100 s along with size exclusion chromatography and dynamic light scattering of refolding samples showed that the overall refolding yield decreased as bhx was unfolded for longer periods of time. Furthermore, the losses resulted from aggregate formation during refolding. This suggests that a change occurs over time in the unfolded or denatured state ensemble that increases the propensity for aggregation upon the shift to more native-favoring conditions. Alternatively aggregate nuclei may be able to form even under high denaturant conditions, and these subsequently grow and consume monomer when placed under native-favoring conditions.
机译：β-螺旋在许多病原体中是重要的蛋白质折叠，并且是折叠路径预测的具有挑战性的系统，因为它主要是通过沿主要序列的非局部相互作用来稳定的。这种折叠的有用的实验模型是P22尾钉蛋白（β-螺旋结构域（bhx））的单体截短。该报告描述了bhx的化学变性和复性的系统化体外研究。平衡化学变性实验的结果与二态折叠机制一致，但仅显示部分可逆性。停止流荧光研究显示单个展开步骤，但是两个重折叠步骤。缓慢的重折叠步骤可能部分归因于脯氨酸异构化，这是基于在PPIase存在下重折叠过程中速率增加以及该步骤的相对幅度增加，并且在两次跳跃重折叠实验中观察到的延迟时间增加了5-100 s。 。然而，延迟时间大于100 s的两次跳跃重折叠实验，以及尺寸排阻色谱和重折叠样品的动态光散射显示，随着bhx的展开时间延长，整体重折叠产量降低。此外，损失是由于重新折叠过程中形成骨料造成的。这表明，在未折叠或变性状态的集合中，随着时间的推移会发生变化，这会增加向自然条件迁移后聚集的倾向。可选择地，即使在高变性条件下，聚集核也可能形成，并且当置于天然条件下时，这些聚集核随后生长并消耗单体。

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