Nanopore technique shows transport mechanism of chaperone proteins at single-molecule level

Proteins control most functions in the body and their dysfunction can have serious consequences, such as neurodegenerative diseases or cancers. Therefore, cells have mechanisms to control protein quality.

In animal and human cells, Hsp70 class chaperones are at the heart of this control system, overseeing a wide range of biological processes. Yet, despite their crucial role, the precise molecular mechanism of Hsp70 chaperones has remained elusive for decades.

Using a cutting-edge monomolecular nanopore technique, a team from the University of Geneva (UNIGE), in collaboration with EPFL, has made a significant advance in determining how Hsp70 chaperones generate the force necessary to manipulate the structure of their client proteins. . These results, which put an end to a decade of debate, are published in Natural communications.

Proteins must fold into specific three-dimensional shapes to function properly. Among their many roles, chaperone proteins like Hsp70 generally help with proper protein folding. To carry out these tasks, Hsp70s must forcefully manipulate protein structure, extracting them from spontaneously formed aggregates or facilitating protein translocation into key cellular compartments, such as mitochondria.

In this context, during the 1990s and early 2000s, intense debate took place on the mechanism by which Hsp70 chaperones drive protein translocation, with two main models proposed based on different sets of experiments. , but without a definitive answer.

In 2006, a new theory, called Entropic Pulling, was proposed by Professor Paolo De Los Rios of EPFL and Professor Pierre Goloubinoff of the University of Lausanne (UNIL) and their collaborators. Entropic Pulling could explain all existing observations of protein translocation in mitochondria and could also be applied to other cellular functions of Hsp70s, such as protein disaggregation.

Experimental evidence

Over the years, this theory has allowed the interpretation of an increasing number of results but has remained without direct experimental confirmation.

The group of Chan Cao, new assistant professor in the Department of Inorganic and Analytical Chemistry of the Faculty of Sciences of UNIGE, specializes in the bioanalysis of single molecules, in particular the detection of nanopores. This innovative approach involves reading the ionic current response as a single molecule passing through a nanoscale pore, which can be either an assembly of biological proteins embedded in a lipid membrane or a manufactured solid material.

The development of nanopore technology aims to create high-resolution sensors to detect target molecules within complex matrices and to sequence biopolymers.

In recent work, the team leveraged nanopore technology to mimic the in vivo pattern of protein translocation at the single-molecule level. Professor Chan Cao explained: “Our results provide clear evidence for the entropic traction mechanism of Hsp70 chaperones, excluding the other two previously proposed models, namely Power Stroke and Brownian Ratchet. »

A powerful force at the molecular level

In the entropic pulling mechanism, the chaperone, by pulling on the target protein, increases its range of motion, generating what is called an entropic force. Verena Rukes, Ph.D. student and lead author of the study, explains: “Our analysis estimated the force of entropic traction to be approximately 46 pN over distances of 1 nm, indicating a remarkably strong force at the molecular level.”

Professor Paolo De Los Rios from the Institute of Physics and the Institute of Bioengineering at EPFL said: “Our theory proposed in 2006 accounted for the essential physics of the system comprising Hsp70, the translocation protein and the translocation pore, but ultimately it remains a theory, even if it is indirectly in agreement with most observations.

“Thanks to the magnificent work of Professor Chan Cao and his team, we now have direct evidence of this and, most importantly, a quantitative estimate of its strength, which turns out to be remarkably high, further explaining why Hsp70s are so efficient in modifying the structure of their target proteins.

Importantly, this research establishes nanopore approaches as a powerful single-molecule technique for exploring molecular mechanisms of protein action.