Single-molecule evidence of Entropic Pulling by Hsp70 chaperones

Abstract

Hsp70 chaperones are central components of the cellular network that ensure the structural quality of proteins. Despite their crucial roles in processes as diverse as the prevention of protein aggregation and protein translocation into organelles, their molecular mechanism of action has remained a hotly debated issue. Due to a lack of suitable methods, no experimental data has directly proven any of the models that have been proposed (Power Stroke, Brownian Ratchet, and Entropic Pulling). Recently, nanopores have emerged as a powerful tool to analyze the function of motor enzymes, as well as protein-protein interactions. Here, we used an in vitro single-molecule nanopore to mimic in vivo translocation of proteins, and to investigate the molecular mechanism of Hsp70. Our experiments demonstrate that Hsp70s forcefully extract polypeptide substrates that are trapped inside the pore. The forces they exert are strong at the molecular level, being equivalent to 46 pN over distances of 1 nm, and depend on the size of Hsp70. These findings provide unambiguous evidence supporting the Entropic Pulling mechanism of action of Hsp70s, thus solving a long-standing debate, and proposing a potentially universal principle governing diverse cellular processes. In addition, these results emphasize the utility of biological nanopores for studying protein function at the single-molecule level.