The mechanochemistry of copper reports on the directionality of unfolding in model cupredoxin proteins.
The mechanochemistry of copper reports on the directionality of unfolding in model cupredoxin proteins.
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DOI：
10.1038/ncomms8894
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发表时间：
2015-08-03
影响因子：
16.6
通讯作者：
Garcia-Manyes S
中科院分区：
文献类型：
--
作者：
Beedle AEM;Lezamiz A;Stirnemann G;Garcia-Manyes S
了解蛋白质展开的方向性和顺序对于阐明潜在的折叠自由能格局至关重要。在金属蛋白中增加了一层额外的复杂性，其中金属辅因子参与了蛋白质正确的功能折叠。然而，有机金属相互作用在折叠(非)折叠时被动态破坏和重塑的确切机制在很大程度上是未知的。在这里，我们使用单分子力谱、原子力显微镜结合蛋白质工程和分子动力学模拟来研究蓝铜蛋白、天青蛋白和藻蓝蛋白各自的展开途径。利用天然铜中心的纳米力学性质作为结构嵌入的分子报告，我们证明了这两种蛋白质都是通过两条独立的、竞争的途径展开的。我们的结果为一种新的动力学分配场景提供了实验证据，在该场景中，蛋白质可以通过位于N和C末端的两个不同的主要过渡态随机展开，这两个过渡态决定了展开发生的方向。在金属蛋白中，金属辅因子参与正确折叠的形成。在这里，作者使用单分子力谱和天然铜中心作为嵌入的内部报告，证明了蓝铜蛋白天青素和藻蓝蛋白在力的作用下通过两条独立的竞争路径展开。
Understanding the directionality and sequence of protein unfolding is crucial to elucidate the underlying folding free energy landscape. An extra layer of complexity is added in metalloproteins, where a metal cofactor participates in the correct, functional fold of the protein. However, the precise mechanisms by which organometallic interactions are dynamically broken and reformed on (un)folding are largely unknown. Here we use single molecule force spectroscopy AFM combined with protein engineering and MD simulations to study the individual unfolding pathways of the blue-copper proteins azurin and plastocyanin. Using the nanomechanical properties of the native copper centre as a structurally embedded molecular reporter, we demonstrate that both proteins unfold via two independent, competing pathways. Our results provide experimental evidence of a novel kinetic partitioning scenario whereby the protein can stochastically unfold through two distinct main transition states placed at the N and C termini that dictate the direction in which unfolding occurs. In metalloproteins, a metal cofactor participates in the formation of the correct fold. Here the authors demonstrate—using single molecule force spectroscopy and the native copper centre as an embedded internal reporter—that the blue-copper proteins azurin and plastocyanin unfold via two independent competing pathways under force.