Piotr E. Marszałek

ABSTRACT DnaK, a prokaryotic Hsp70 chaperone, plays a central role in proteostasis by restoring native structures to heat-denatured proteins in an ATP-hydrolysis–dependent manner. While structures of DnaK in complex with nucleotides, co-chaperones, and short peptides have been resolved, structures with larger, stably folded substrates—such as firefly luciferase (Fluc, 61 kDa)—are lacking, limiting mechanistic understanding of how DnaK refolds such proteins. Here, we generated models of the DnaK–Fluc complex using AlphaFold3 and evaluated their mechanistic relevance. In one of three major model clusters, Fluc is unexpectedly immobilized beneath the DnaK α-helical lid against the nucleotide-binding domain (NBD), rather than interacting primarily with the substrate-binding domain β (SBDβ), as commonly assumed. All-atom molecular dynamics simulations indicate that, in this configuration, the lid can engage a thermally destabilized Fluc helix (residues 405–411), which we recently identified as the first—and likely the only—helix to irreversibly melt at 42 °C. Upon binding, the lid forms extensive hydrogen-bonding interactions with the melted helix. These interactions persist during lid movement toward SBDβ (following ATP hydrolysis), enabling the lid to actively extract the helix from the Fluc surface. In contrast, simulations with the helix in its native folded state show that the lid cannot extract it, leaving the native structure unaffected. Equilibrium simulations further indicate that, once extracted and mechanically stretched, the melted helix can refold to its native conformation. Together, these findings suggest a revised mechanism for DnaK-mediated protein refolding, in which the α-helical lid selectively recognizes structurally compromised segments, forms stabilizing hydrogen bonds, and—powered by ATP hydrolysis—mechanically pulls them away from the protein surface to facilitate their refolding. SIGNIFICANCE DnaK is a model chaperone, which can reactivate thermally denatured proteins. Over the span of 40 years, significant findings have been made about DnaK’s structure, dynamics and interactions with its co-chaperones, the exact molecular mechanism by which DnaK refolds misfolded proteins remains a mystery. This work exploited Alphafold3 to generate atomistic models of complexes between DnaK and Firefly luciferase. Molecular dynamics simulations directly captured how DnaK may assist thermally denatured proteins by mechanically pulling out their misfolded helices. This study provides a new insight into the DnaK mechanism.

Despite its ubiquity in nature, some details of the animal olfactory system remain unclear. One such mystery is the mechanism by which olfactory receptors (ORs) recognize the olfactant molecules they bind to. Some evidence indicates that ORs can distinguish between molecules that differ only in isotopic composition, suggesting that olfactants' vibrational modes may play a role in their recognition. In 2023, the first experimental structure of a human olfactory receptor—OR51E2—was produced, providing computational scientists an opportunity to shed additional light on this problem. We compute the infrared spectrum of the olfactant propionate (C2H5COO–) in the OR51E2 binding site by quantum mechanics/molecular mechanics, with atomic positions taken at 25 time points over a 500 ns molecular dynamics simulation. By comparing the spectra for all 32 possible hydrogen/deuterium isotopic combinations in propionate and across the time snapshots, we estimate the relative strength of the isotope effects and thermal fluctuations in the vibrational energy of C2H5COO–. The high-frequency C–H modes are are about 800 cm^{-1} higher in energy than their deuterated counterparts, a large separation relative both to their fluctuations over time and to the thermal energy available at physiological temperature. Lower-frequency vibrations do not display such a clear isotopic separation. Thus, any vibrational component to olfactant recognition—especially one that allows distinguishing between isotopes—is likely to involve these high-frequency modes.

Despite its ubiquity in nature, some details of the animal olfactory system remain unclear. One such mystery is the mechanism by which olfactory receptors (ORs) recognize the olfactant molecules they bind to. Some evidence indicates that ORs can distinguish between molecules that differ only in isotopic composition, suggesting that olfactants' vibrational modes may play a role in their recognition. In 2023, the first experimental structure of a human olfactory receptor—OR51E2—was produced, providing computational scientists an opportunity to shed additional light on this problem. We compute the infrared spectrum of the olfactant propionate (C2H5COO–) in the OR51E2 binding site by quantum mechanics/molecular mechanics, with atomic positions taken at 25 time points over a 500 ns molecular dynamics simulation. By comparing the spectra for all 32 possible hydrogen/deuterium isotopic combinations in propionate and across the time snapshots, we estimate the relative strength of the isotope effects and thermal fluctuations in the vibrational energy of C2H5COO–. The high-frequency C–H modes are are about 800 cm^{-1} higher in energy than their deuterated counterparts, a large separation relative both to their fluctuations over time and to the thermal energy available at physiological temperature. Lower-frequency vibrations do not display such a clear isotopic separation. Thus, any vibrational component to olfactant recognition—especially one that allows distinguishing between isotopes—is likely to involve these high-frequency modes.

Chaperones are a large family of proteins crucial for maintaining cellular protein homeostasis. One such chaperone is the 70 kDa heat shock protein (Hsp70), which plays a crucial role in protein (re)folding, stability, functionality, and translocation. While the key events in the Hsp70 chaperone cycle are well established, a relatively small number of distinct substrates were repetitively investigated. This is despite Hsp70 engaging with a plethora of cellular proteins of various structural properties and folding pathways. Here we analyzed novel Hsp70 substrates, based on tandem repeats of NanoLuc (Nluc), a small and highly bioluminescent protein with unique structural characteristics. In previous mechanical unfolding and refolding studies, we have identified interesting misfolding propensities of these Nluc-based tandem repeats. In this study, we further investigate these properties through in vitro bulk experiments. Similar to monomeric Nluc, engineered Nluc dyads and triads proved to be highly bioluminescent. Using the bioluminescence signal as the proxy for their structural integrity, we determined that heat-denatured Nluc dyads and triads can be efficiently refolded by the E. coli Hsp70 chaperone system, which comprises DnaK, DnaJ, and GrpE. In contrast to previous studies with other substrates, we observed that Nluc repeats can be efficiently refolded by DnaK and DnaJ, even in the absence of GrpE co-chaperone. Taken together, our study offers a new powerful substrate for chaperone research and raises intriguing questions about the Hsp70 mechanisms, particularly in the context of structurally diverse proteins.

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.