Opposing Effects of Periplasmic Chaperones on Protein Folding.

Abstract

Protein translocation across the bacterial SecYEG channel involves mechanical constraints arising from ATP-driven SecA activity, geometric confinement within the translocon, and folding of the emerging polypeptide on the periplasmic side. Periplasmic chaperones assist substrate maturation during this process, but how they influence protein folding under force remains poorly understood. Using protein L as a model two-state substrate, we applied physiologically relevant force pulses using custom-built single-molecule magnetic tweezers to examine how bacterial periplasmic chaperones modulate folding under tension. To isolate the direct effects of individual chaperones, these experiments were performed in the absence of SecA and the SecYEG translocon. We show that the periplasmic chaperones PpiD and DsbC increase folding probability and accelerate refolding under force, while having minimal effect on unfolding kinetics. In contrast, Spy and Skp reduce folding probability and suppress refolding, consistent with holdase-like behaviour. These observations show that distinct classes of periplasmic chaperones differentially modulate folding probability and refolding kinetics under mechanical force. Increased folding probability correspondingly enhances the expected mechanical work output of substrate folding under force. Together, our findings establish a quantitative framework for investigating how bacterial periplasmic chaperones modulate protein folding under controlled mechanical conditions.