Steered Molecular Dynamics (SMD) has been seen to provide the potential of mean force (PMF) along a peptide unfolding pathway effectively but at significant computational cost, particularly in all-atom solvents. Adaptive steered molecular dynamics (ASMD) has been seen to provide a significant computational advantage by limiting the spread of the trajectories in a staged approach. The contraction of the trajectories at the end of each stage can be performed by taking a structure whose nonequilibrium work is closest to the Jarzynski average (in naive ASMD) or by relaxing the trajectories under a no-work condition (in full-relaxation ASMD—namely, FR-ASMD). Both approaches have been used to determine the energetics and hydrogen-bonding structure along the pathway for unfolding of a benchmark peptide initially constrained as an α-helix in a water environment. The energetics are quite different to those in vacuum, but are found to be similar between implicit and explicit solvents. Surprisingly, the hydrogen-bonding pathways are also similar in the implicit and explicit solvents despite the fact that the solvent contact plays an important role in opening the helix.

The regulation of protein function through ligand-induced conformational changes is crucial for many signal transduction processes. The binding of a ligand alters the delicate energy balance within the protein structure, eventually leading to such conformational changes. In this study, we elucidate the energetic and mechanical changes within the subdomains of the nucleotide binding domain (NBD) of the heat shock protein of 70 kDa (Hsp70) chaperone DnaK upon nucleotide binding. In an integrated approach using single molecule optical tweezer experiments, loop insertions, and steered coarse-grained molecular simulations, we find that the C-terminal helix of the NBD is the major determinant of mechanical stability, acting as a glue between the two lobes. After helix unraveling, the relative stability of the two separated lobes is regulated by ATP/ADP binding. We find that the nucleotide stays strongly bound to lobe II, thus reversing the mechanical hierarchy between the two lobes. Our results offer general insights into the nucleotide-induced signal transduction within members of the actin/sugar kinase superfamily.ATPase | laser trapping | elasticity | force | protein extension

Microtubules (MTs) are structural components essential for cell morphology and organization. It has recently been shown that defects in the filament’s lattice structure can be healed to create stronger filaments in a local area and ultimately cause global changes in MT organization and cell mobility. The ability to break, causing a defect, and heal appears to be a physiologically relevant and important feature of the MT structure. Defects can be created by MT severing enzymes and are target sites for complete severing or for healing by newly incorporated dimers. One particular lattice defect, the MT lattice ‘‘seam” interface, is a location often speculated to be a weak site, a site of disassembly, or a target site for MT binding proteins. Despite seams existing in many MT structures, very little is known about the seam’s role in MT function and dynamics. In this study, we probed the mechanical stability of the seam interface by applying coarse-grained indenting molecular dynamics. We found that the seam interface is as structurally robust as the typical lattice structure of MTs. Our results suggest that, unlike prior results that claim the seam is a weak site, it is just as strong as any other location on the MT, corroborating recent mechanical measurements.

Owing to the cooperativity of protein structures, it is often almost impossible to identify independent subunits, flexible regions, or hinges simply by visual inspection of static snapshots. Here, we use single-molecule force experiments and simulations to apply tension across the substrate binding domain (SBD) of heat shock protein 70 (Hsp70) to pinpoint mechanical units and flexible hinges. The SBD consists of two nanomechanical units matching 3D structural parts, called the α-and β-subdomain. We identified a flexible region within the rigid β-subdomain that gives way under load, thus opening up the α/β interface. In exactly this region, structural changes occur in the ATP-induced opening of Hsp70 to allow substrate exchange. Our results show that the SBD's ability to undergo large conformational changes is already encoded by passive mechanics of the individual elements.laser trapping | parallel pathways | elasticity | force | protein extension W hen looking at protein structures at atomic resolution, it is often tempting to use macroscopic mechanical analogies to describe their function as molecular machines. However, such analogies are often misleading because boundaries between independently stable subdomains cannot often be determined from structures, owing to the high cooperativity of protein folding and structural transitions. Single-molecule protein nanomechanics have emerged as a tool to force biomolecules through their conformational space and, hence, identify hinges, breaking points, and mechanically stable subdomains (1-3).A prominent example of a protein machine undergoing large conformational change during its functional cycle is the ATP-regulated Hsp70 chaperone DnaK-a central molecular chaperone of the protein quality control network in a cell (4-6). Once ATP is bound to the nucleotide binding domain (NBD, blue-yellow; Fig. 1A) of DnaK, the initially closed substrate binding domain (SBD) opens its binding cleft by engaging the β-subdomain to the NBD. In doing so, it undergoes a dramatic ∼10 Å displacement of its lid subdomain ( Fig. 1A; refs. 7 and 8) to allow exchange of substrates (9). Several crystal structures of the isolated SBD (in which the NBD is absent) have been solved (10-15). In these structures, the absence or presence of peptide clients or nonnatural ligands induce no significant structural changes in the closed conformation. There is no indication in the crystal structures of the huge conformational change of the lid domain of the SBD, seen in the ATP form of the full-length two-domain DnaK. Therefore, the large conformational change of the SBD is only observed in the two-domain DnaK after ATP binding. Thus, although the crystal structures provide us with valuable insights into the 3D arrangement of individual atoms, the thermodynamic and mechanical stability of individual substructures are difficult to predict based on this information alone. Here, we ask how the large ATP-induced changes of the SBD, as seen in the two-domain DnaK, are mirrored in the subdomain integrity and nano...