Elasticity and mechanical metamaterials
How does pulling force change how chaperones help proteins refold?
Open access · cc by · source: Europe PMC
How hard a stretched protein is pulled decides how strongly the helper protein DnaJ grabs it, and the full chaperone team turns from blocking folding to speeding it up.
Study at a glance
- Design
- Animal / in-vitro — Single-molecule force-clamp atomic force spectroscopy (force-quench protocols) on polyproteins with added chaperones, plus explicit-solvent MD and a kinetic binding model
- N
- No single N; refolding yields pooled over many single-molecule trajectories per condition, with errors from bootstrapping.
- Population
- Purified polyproteins of ubiquitin (9 repeats), titin I27 (8 repeats) and titin Z1 (8 repeats) with E. coli chaperones DnaJ, DnaK and GrpE
- Outcome
- Refolding efficiency and folding rate after mechanical unfolding, as a function of quench time, time held extended and stretching force
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Key findings
Alone, each chaperone cut ubiquitin's refolding from about 75% to about 30%, acting as a 'holdase'. DnaJ bound the stretched ubiquitin chain, apparently via a specific sequence motif, whereas DnaK bound only the partly collapsed intermediates. DnaJ binding depended non-monotonically on force: refolding fell to about 10% at intermediate forces but was higher at 50 and 300 pN. The complete chaperone system instead sped up ubiquitin folding (rate 0.92 versus 0.52 per second) and raised Z1 refolding from about 15% to about 35%.
Methodology
Using an atomic force microscope in force-clamp mode, the researchers unfolded chains of identical protein domains by pulling on them, relaxed the force to let them refold, then pulled again to count how many domains had regained their mechanical stability. They repeated this with chaperones DnaJ or DnaK alone, or the complete DnaK-DnaJ-GrpE system with ATP, varying how long the protein was held stretched and how hard it was pulled. Molecular dynamics simulations and a kinetic model were used to explain the force dependence and extract binding constants.
Limitations
Sequence specificity of DnaJ is inferred by comparing two different proteins, because ubiquitin mutants in the motif failed to fold, so the motif was never directly tested. Experiments use artificial tandem polyproteins stuck to a gold surface at fixed chaperone concentrations, so relevance to forces inside cells is speculative. The simulation explanation of force-dependent binding is qualitative, and binding constants come from fitting a multi-parameter kinetic model.
How this study connects
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