Department of Inorganic and Analytical Chemistry, Chemistry and Biochemistry, University of Geneva, Geneva, 1211, Switzerland.
Global Health Institute, School of Life Sciences, École Polytechnique Fédérale de Lausanne - EPFL, Lausanne, 1015, Switzerland.
Nat Commun. 2024 Oct 8;15(1):8604. doi: 10.1038/s41467-024-52674-y.
Hsp70 chaperones are central components of the cellular network that ensures the structural quality of proteins. Despite crucial roles in processes such as protein disaggregation and protein translocation into organelles, their physical mechanism of action has remained hotly debated. To the best of our knowledge, no experimental data has directly proven any of the models proposed to date (Power Stroke, Brownian Ratchet, or Entropic Pulling) due to a lack of suitable methods. Here, we use nanopores, a powerful single-molecule tool, to investigate the mechanism of Hsp70s. We demonstrate that Hsp70s extract trapped polypeptide substrates from the nanopore by generating strong forces (equivalent to 46 pN over distances of 1 nm), that rely on the size of Hsp70. The findings provide unambiguous evidence of the Entropic Pulling mechanism, thus solving a long-standing debate, and proposing a potentially universal principle governing diverse cellular processes. Additionally, these results highlight the utility of biological nanopores for protein studies.
热休克蛋白 70 伴侣是确保蛋白质结构质量的细胞网络的核心组成部分。尽管它们在蛋白质解聚和蛋白质向细胞器易位等过程中起着至关重要的作用，但它们的物理作用机制仍存在激烈的争论。据我们所知，由于缺乏合适的方法，迄今为止没有任何实验数据直接证明任何已提出的模型（Power Stroke、Brownian Ratchet 或 Entropic Pulling）。在这里，我们使用纳米孔这一强大的单分子工具来研究 Hsp70 的机制。我们证明 Hsp70 通过产生强大的力（在 1nm 的距离上相当于 46pN）从纳米孔中提取被困的多肽底物，这依赖于 Hsp70 的大小。这些发现提供了明确的证据证明了 Entropic Pulling 机制，从而解决了长期存在的争议，并提出了一个潜在的普遍原则，用于控制多种细胞过程。此外，这些结果突出了生物纳米孔在蛋白质研究中的应用。
