Zhang Hongyu, Jackson Sophie E
Yusuf Hamied Department of Chemistry, University of Cambridge, Cambridge, UK.
Protein Sci. 2025 Mar;34(3):e70048. doi: 10.1002/pro.70048.
It is hard to imagine how proteins can thread and form knots in their polypeptide chains, but they do. These topologically complex structures have challenged the traditional protein folding views of simple funnel-shaped energy landscapes. Previous experimental studies on the folding mechanisms of deeply knotted proteins with a single trefoil knot have yielded evidence that this topology has a more complicated folding landscape than other simpler proteins. However, to date, there have been no attempts to study the folding of any protein in which multiple threading events are needed to create more than one knot within a single polypeptide chain. Here, we report the construction and characterization of an artificial tandemly knotted protein. We find compelling evidence that both domains of the protein form trefoil knots with similar structures and stabilities to the parent single trefoil-knotted protein. In addition, we show that this tandemly knotted protein has a complex folding pathway in which there are additional very slow folding phases that we propose correspond to the formation of the second knot within the system. We also find evidence that during folding this protein gets transiently trapped in deep kinetic traps, however, the majority of protein chains (>90%) manage to partially unfold and acquire the native tandem-knot topology. This work highlights the fact that Nature can tolerate more complex protein topologies than we thought, and despite considerable misfolding during folding, protein chains can find their way to the native state even in the absence of molecular chaperones.
很难想象蛋白质如何在其多肽链中穿线并形成结，但它们确实能做到。这些拓扑结构复杂的蛋白质对传统的简单漏斗状能量景观的蛋白质折叠观点提出了挑战。先前对具有单个三叶结的深度打结蛋白质折叠机制的实验研究表明，这种拓扑结构的折叠景观比其他更简单的蛋白质更为复杂。然而，迄今为止，还没有人尝试研究在单个多肽链中需要多次穿线事件才能形成多个结的任何蛋白质的折叠情况。在此，我们报告了一种人工串联打结蛋白质的构建和表征。我们发现了令人信服的证据，表明该蛋白质的两个结构域都形成了与亲本单三叶结蛋白质结构和稳定性相似的三叶结。此外，我们表明这种串联打结蛋白质具有复杂的折叠途径，其中存在额外的非常缓慢的折叠阶段，我们认为这与系统中第二个结的形成相对应。我们还发现有证据表明，在折叠过程中，这种蛋白质会暂时被困在深度动力学陷阱中，然而，大多数蛋白质链（>90%）设法部分展开并获得天然的串联结拓扑结构。这项工作突出了这样一个事实，即自然界能够容忍比我们想象中更复杂的蛋白质拓扑结构，并且尽管在折叠过程中存在大量错误折叠，但即使在没有分子伴侣的情况下，蛋白质链也能找到通往天然状态的途径。
