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a) Abstract DnaK is a prokaryotic Hsp70 chaperone, with numerous functions in helping to fold nascent polypeptides and more generally in proteostasis. It also restores native structures to heat-shocked proteins in an ATP-hydrolysis-dependent manner. The structures of DnaK complexes with nucleotides, co-chaperones andsmallpeptides have already been resolved. However, there are no structures of DnaK complexes with larger, mostly folded substrates, such as firefly luciferase (Fluc, 61 kDa), which impedes the understanding of the mechanism through which DnaK refolds such large proteins. Here, we generated a model of a DnaK-firefly luciferase complex with Alphafold3, and examined its dynamics with all-atom molecular dynamics simulations. In this complex, Fluc is immobilized under the DnaK alpha-helical lid against the NBD, not the SBDβ, contrary to the data reported in the literature for model peptides. The DnaK lid is positioned strategically over Fluc’s helix 405-411, which we recently determined to be the first (and likely the only) helix melted in Fluc at 42 °C. We simulated the interaction between DnaK and the helix in its native and misfolded state and found that during the lid translocation toward the SBDβ, only the melted helix follows the lid and is actively pulled out from Fluc, while the native helix is not dislocated. These observations suggest a new model for the DnaK chaperone mechanism, where the alpha helical lid forms hydrogen bonds to the protein segment to be structurally tested. Lid pulls out only highly deformable misfolded helices, allowing them to refold into their native structures, and does not pull out those that are correctly folded because they are not deformable. Broader Audience Statementc) DnaK is a model chaperone, which can reactivate thermally denatured proteins. Even though a plethora of significant findings about DnaK structure, dynamics and interactions with its co-chaperone have been accumulated over 30 years, the exact molecular mechanism by which DnaK refolds misfolded proteins remains a mystery. This work exploited the ability of the Alphafold3 platform to generate an atomistic model for a complex between DnaK and Firefly luciferase and used molecular dynamics simulations to directly capture how DnaK may assist denatured proteins by mechanically pulling out their misfolded helices. This study provides a new insight into the DnaK mechanism. 

Protein self-assembly plays a vital role in a myriad of biological functions and in the construction of biomaterials. Although the physical association underlying these assemblies offers high specificity, the advantage often compromises the overall durability of protein complexes. To address this challenge, we propose a novel strategy that reinforces the molecular self-assembly of protein complexes mediated by their ligand. Known for their robust noncovalent interactions with biotin, streptavidin (SAv) tetramers are examined to understand how the ligand influences the mechanical strength of protein complexes at the nanoscale and macroscale, employing atomic force microscopy-based single-molecule force spectroscopy, rheology, and bioerosion analysis. Our study reveals that biotin binding enhances the mechanical strength of individual SAv tetramers at the nanoscale. This enhancement translates into improved shear elasticity and reduced bioerosion rates when SAv tetramers are utilized as cross-linking junctions within hydrogel. This approach, which enhances the mechanical strength of protein-based materials without compromising specificity, is expected to open new avenues for advanced biotechnological applications, including self-assembled, robust biomimetic scaffolds and soft robotics.