Microtubules (MTs) are made of αβ-tubulin heterodimers joined longitudinally into protofilaments (PFs). PFs associate laterally to form the hollow cylinder of an MT. Most MTs in cells contain 13 PFs, however the most common in vitro MTs have 13 or/and 14 PFs. The B-lattice (where two adjacent PFs are laterally connected by interactions between the same type of tubulin monomers: α-α/β-β) is dominant in the MT cylinder structure. Additionally, each MT cylinder contains a single lateral interaction created by the A-lattice (where two adjacent PFs are connected through α-β and β-α interactions) and called the seam. The proper functioning of MTs has a key role in the process of cell division, cell motility and cell development due to the fact that MTs are responsible for cell morphology, cell interior organization and intracellular cargo transport. Dynamic MTs rearrangements by breaking and healing are crucial for accomplishing and maintaining their cellular functions. MT breakage can occur through one of two mechanisms: breaking from high frequency buckling of PFs and breaking induced by microtubule-severing enzymes. Due to the high complexity of MTs, experimental techniques are not able to fully answer the question which of the A/B MT lattices is the "sweet spot" for the binding of microtubule-severing enzymes. Therefore, a combination of experimental and theoretical techniques is required to solve this problem. We use computational methods to probe and compare the mechanical stability of the both MTs lattices using coarse-grained indenting molecular dynamics. With the knowledge of how the B-lattice behaves (with/without defects) we can compare its mechanical stability with that of the A-lattice. Furthermore, for both lattices computationally predicted breaking forces, bending angles distributions and MTs length factors are comparable with experimental data obtained from in vitro severing assays and AFM experiments.

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.

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.

The data files are labeled according to figure numbers in the associated Open Access article, H. R. Bureau, D. Merz Jr., E. Hershkovits, S. Quirk and Rigoberto Hernandez, Constrained unfolding of a helical peptide: Implicit versus Explicit Solvents, PLoS ONE 10, e0127034 (2015). (doi: 10.1371/journal.pone.0127034). An in-depth description of the content of the data files can be found in ReadMe_ASMD1Data_20150526.