Proteins are essential for most bodily functions, and if they fail to work correctly, it can lead to serious health issues like neurodegenerative diseases or cancer. As a result, cells have developed methods to maintain protein quality. In both animal and human cells, Hsp70 chaperones play a central role in this quality control system, managing various biological activities. However, the exact molecular mechanisms behind Hsp70 chaperones have been unclear for years. Recently, a research team from the University of Geneva (UNIGE) and EPFL has made an important discovery using advanced nanopore single-molecule techniques to understand how Hsp70 chaperones create the necessary force to adjust the structure of their target proteins. Their findings, which clarify a long-standing debate, are detailed in Nature Communications.
For proteins to function correctly, they must fold into specific three-dimensional shapes. One of the main roles of chaperone proteins like Hsp70s is to assist in this proper folding. To effectively perform these roles, Hsp70s need to actively reshape proteins, either by removing them from aggregates that form spontaneously or by aiding in their transport into vital cell compartments, such as mitochondria.
In the 1990s and early 2000s, there was significant debate regarding how Hsp70 chaperones facilitate protein transport, with two primary models emerging from various experiments, yet no conclusive answers were found. In 2006, a new theory called Entropic Pulling was introduced by Prof. Paolo De Los Rios at EPFL and Prof. Pierre Goloubinoff at the University of Lausanne (UNIL) along with their team. This theory could account for all previous observations regarding mitochondrial protein transport and could also extend to other functions of Hsp70s, such as protein disassembly.
Experimental Support
While this theory has interpreted an increasing amount of data over the years, direct experimental confirmation was still lacking. The Chan Cao group, which includes a new assistant professor in the Department of Inorganic and Analytical Chemistry at UNIGE Faculty of Science, focuses on single-molecule bioanalysis, specifically employing nanopore detection. This innovative technique measures the ionic current response as individual molecules pass through a tiny pore, which might be a natural protein assembly in a lipid membrane or a manufactured solid-state structure. The aim of developing nanopore technology is to create high-resolution sensors capable of detecting specific molecules within complex mixtures and for sequencing biopolymers.
In their latest study, the team applied nanopore technology to replicate the protein transport process in a living organism at the single-molecule level. Prof. Chan Cao remarked, “our findings provide solid evidence for the Entropic Pulling mechanism of Hsp70 chaperones, effectively dismissing the other two previously proposed models: Power Stroke and Brownian Ratchet.”
A Forceful Discovery
Through the Entropic Pulling mechanism, the chaperone increases the movement range of the target protein by exerting a pull, which generates what is known as an entropic force. Study lead author Verena Rukes, a PhD student, explains, “our analysis estimated the strength of Entropic Pulling to be around 46 pN over a distance of 1 nm, indicating an exceptionally strong force at the molecular level.”
Prof. Paolo De Los Rios from EPFL’s Institute of Physics and Institute of Bioengineering states, “our 2006 theory accounted for much of the physics involved in the Hsp70 system, which includes the translocating protein and the pore. Still, it remained a theoretical framework, indirectly correlating with most observations. Thanks to the exceptional work from Prof. Chan Cao and her team, we now have direct evidence of our theory and, importantly, a quantitative measure of its strength, which is impressively high and helps to explain why Hsp70s are so effective in altering the structure of their target proteins.”
This research highlights the potential of nanopore methods as a robust technique for investigating the molecular mechanisms of protein functions.
