The investigation and understanding of the folding mechanism of multidomain proteins is still a challenge in structural biology. The use of single-molecule Förster resonance energy transfer offers a unique tool to map conformational changes within the protein structure. Here, we present a study following denaturant-induced unfolding transitions of yeast phosphoglycerate kinase by mapping several inter- and intradomain distances of this two-domain protein, exhibiting a quite heterogeneous behavior. On the one hand, the development of the interdomain distance during the unfolding transition suggests a classical two-state unfolding behavior. On the other hand, the behavior of some intradomain distances indicates the formation of a compact and transient molten globule intermediate state. Furthermore, different intradomain distances measured within the same domain show pronounced differences in their unfolding behavior, underlining the fact that the choice of dye attachment positions within the polypeptide chain has a substantial impact on which unfolding properties are observed by single-molecule Förster resonance energy transfer measurements. Our results suggest that, to fully characterize the complex folding and unfolding mechanism of multidomain proteins, it is necessary to monitor multiple intra- and interdomain distances because a single reporter can lead to a misleading, partial, or oversimplified interpretation.

中文翻译：

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在多域蛋白质中绘制多个距离以识别折叠中间体

多域蛋白折叠机制的研究和理解仍然是结构生物学中的一个挑战。使用单分子 Förster 共振能量转移提供了一种独特的工具来绘制蛋白质结构内的构象变化。在这里，我们通过绘制这种双域蛋白质的几个域间和域内距离，展示了一种表现出相当异质性的行为，研究了变性剂诱导的酵母磷酸甘油酸激酶的展开转变。一方面，展开过渡期间域间距离的发展表明了经典的二态展开行为。另一方面，一些域内距离的行为表明形成了致密且瞬态的熔球中间态。此外，在同一域内测量的不同域内距离显示出它们展开行为的显着差异，强调了这样一个事实，即多肽链内染料附着位置的选择对通过单分子 Förster 共振能量转移测量观察到的展开特性有重大影响。我们的结果表明，为了充分表征多域蛋白质的复杂折叠和展开机制，有必要监测多个域内和域间距离，因为单个记者可能会导致误导、部分或过度简化的解释。强调了这样一个事实，即多肽链内染料附着位置的选择对通过单分子 Förster 共振能量转移测量观察到的展开特性有重大影响。我们的结果表明，为了充分表征多域蛋白质的复杂折叠和展开机制，有必要监测多个域内和域间距离，因为单个记者可能会导致误导、部分或过度简化的解释。强调了这样一个事实，即多肽链内染料附着位置的选择对通过单分子 Förster 共振能量转移测量观察到的展开特性有重大影响。我们的结果表明，为了充分表征多域蛋白质的复杂折叠和展开机制，有必要监测多个域内和域间距离，因为单个记者可能会导致误导、部分或过度简化的解释。