The 70 kDa Heat Shock Protein (Hsp70) chaperones are critical components of the cell’s protein quality control system, playing key roles in protein folding, translocation, and disaggregation. Proteins synthesized in the cytoplasm rely on Hsp70 chaperones to move through specialized membrane channels/pores into organelles. Despite their well-established functions, the exact physical mechanism by which Hsp70 exerts force on its substrates has remained unresolved.

In a recent study published in Nature Communications, researchers employed nanopore technology and electrophysiology recordings to directly observe the mechanism of Hsp70-mediated pulling at the single-molecule level. Their findings provide the first definitive evidence for the “Entropic Pulling” model, which posits that Hsp70 generates force by reducing entropy when it binds to a substrate emerging from a translocation channel. This entropic reduction prompts the chaperone to move away from the channel, pulling the bound protein with it.

Using the Orbit mini platform for electrophysiology recordings, the team tracked ionic currents through nanopores embedded in artificial lipid membranes. This setup allowed them to monitor the escape of polypeptide substrates under the influence of Hsp70, providing real-time measurements of the chaperone’s pulling force. Remarkably, the researchers observed that Hsp70 could generate forces as strong as 46 pN over a distance of 1 nm, sufficient to extract polypeptides from the nanopore. The force was dependent on the size of Hsp70, in line with the Entropic Pulling model’s predictions, and distinct from other models such as the Power Stroke or Brownian Ratchet.

Furthermore, molecular dynamics simulations supported these findings, revealing that the pulling force scales with the size of the protein domain. The study also showed that this entropic mechanism allows Hsp70 to perform tasks like protein translocation and aggregate disassembly, suggesting a broader application of Entropic Pulling in various cellular processes.

In conclusion, this study establishes Hsp70 as a molecular motor that harnesses entropy to generate force, with implications for cellular protein homeostasis and potential therapeutic interventions in diseases related to protein misfolding and aggregation. The integration of nanopore technology and electrophysiology recordings in this research offers a powerful tool for future investigations into molecular chaperone mechanics.

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Find the full article here: Single-molecule evidence of Entropic Pulling by Hsp70 chaperones

Discover how parallel lipid bilayer recordings can be utilized to study nanopores: https://www.nanion.de/products/orbit-16-tc/