Mechanistic Insights into Periplasmic Chaperones as Regulators of Protein Folding and Translocation in Gram-Negative Bacteria

Abstract

In Gram-negative bacteria, periplasmic chaperones mediate the transport and folding of outer membrane proteins. While their substrate interaction mechanisms are well studied, their functional cycle during periplasmic translocation remains unclear. Using single-molecule force spectroscopy, we demonstrate that SecYEG-associated chaperones PpiD and DsbC act as foldases, delivering up to 45.5 zJ of mechanical work. This activity facilitates substrate extraction from the SecYEG translocation tunnel, reducing ATP consumption during SecA-mediated translocation. In contrast, chaperones such as Spy and Skp function as holdases, stabilizing unfolded substrates to prevent misfolding during translocation. Our findings reveal a unique mechanism by which chaperones adapt to mechanical constraints, optimizing protein folding and translocation in the periplasm.