Aug 2026· Journal of Molecular Biology· Vol 438, pp.
169966
· 0 citations· 58 references
Medicine
TL;DR
It is shown that distinct classes of periplasmic chaperones differentially modulate folding probability and refolding kinetics under mechanical force, and increased folding probability correspondingly enhances the expected mechanical work output of substrate folding under force.
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.
It is shown that short nucleic acids containing Gquadruplex (G4) structure can also catalyze protein folding and uncovers a previously underappreciated role for nucleic acid in proteostasis and offers a new strategy for studying nucleic acid structure-function relationship at residue level.
This work introduces BEAM, a multiscale framework that learns slow collective variables from coarse-grained simulations to guide all-atom enhanced sampling to explain how vectorial secretion accelerates pertactin folding by excluding an off-pathway kinetic trap.
Lan Yang, Qing Luan, Michael C. Baxa et al.· bioRxiv· 0 citations
DegP is a periplasmic protease-chaperone essential for protein quality control and virulence factor trafficking in Gram-negative bacteria. In its apo form, DegP adopts a dynamic ensemble of oligomers derived from trimer building blocks through two competing self-assembly pathways. Upon engaging client proteins, apo DegP oligomers redistribute into discrete cage structures inside which the clients are encapsulated. The cage ensembles formed depend on the size of the bound clients, and notably can include 12mers, 24mers, and 60mers. Previous studies mapped the DegP oligmeric landscape using dynamic light scattering, analytical ultracentrifugation, nuclear magnetic resonance spectroscopy, and electron cryomicroscopy, modalities which in general report ensemble averages and often cannot directly delineate closely related coexisting species. Here, we apply charge detection mass spectrometry (CDMS) to directly measure the masses of individual DegP ions, resolving the complete oligomeric distribution in the absence and presence of four clients of increasing size. We reveal previously undetected odd-numbered oligomers and quantify the relative abundance of each assembly. Through heat-cool cycling CDMS experiments, we track cage distribution changes and reveal client protection and refolding, providing a direct view of the chaperone capabilities of DegP. These results further establish CDMS as a powerful single-molecule tool for dissecting heterogeneous protein assembly landscapes.
Kent R. Vosper, Aizaz Humayun, Matthew S McPartlan et al.· bioRxiv· 0 citations
Mechanosensitive channels of large conductance (MscL) protect bacteria from hypo-osmotic stress by opening a large pore in response to membrane tension. The transmembrane TM1 and TM2 helices together with the N-terminal amphipathic helix function as the primary force-sensing structural domains, whereas the role of the periplasmic loop in the channel gating remains poorly understood. Using experimental and computational approaches, our study demonstrates that mutating the loop residues (A64, Q65, G66, D67) or inserting a four-glycine flexible hinge at D67 site modified the properties of the channel recorded in giant E. coli spheroplasts and liposomes composed of azolectin or negatively charged lipids. Extending the periplasmic loop disrupted mechanical force transmission, reducing channel sensitivity to applied tension. Q65R channel mutant exhibited increased sensitivity in azolectin liposomes that was reduced in negatively charged liposomes and spheroplast membranes. The Q65E mutant showed decreased sensitivity across all preparations tested and exhibited channel flickering in negatively charged liposomes. Molecular dynamics simulations revealed that Q65E produced larger but more structurally restricted conformational changes, whereas wild-type MscL and Q65R exhibited rapid and extensive pore opening over a shorter timeframe. Our findings establish the periplasmic loop as a structural domain fine-tuning MscL gating through electrostatic interactions with surrounding lipids.
K. Duru, Paul Rohde, H. Hafezi et al.· npj Biological Physics and M...· 0 citations
A novel approach is presented that combines pulse metabolic labelling with SILAC and BioID mass spectrometry to temporally resolve protein-protein interactions of a co-chaperone to gain novel insights on how co-chaperone-client interactions may occur over the lifespan of a protein to preserve proteostasis.
It is postulate that Skp in Neisseria must utilize a different mechanism than in E. coli to stabilize substrates by expanding and contracting along its long axis to accommodate substrates of differing sizes.
S. Dubey, J. Stoudenmire, P. Gheinani et al.· Structure· 0 citations
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