Microtubules, the primary components of the chromosome segregation machinery, are stabilized by longitudinal and lateral noncovalent bonds between the tubulin subunits. However, the thermodynamics of these bonds and the microtubule physicochemical properties are poorly understood. Here, we explore the biomechanics of microtubule polymers using multiscale computational modeling and nanoindentations in silico of a contiguous microtubule fragment. A close match between the simulated and experimental force–deformation spectra enabled us to correlate the microtubule biomechanics with dynamic structural transitions at the nanoscale. Our mechanical testing revealed that the compressed MT behaves as a system of rigid elements interconnected through a network of lateral and longitudinal elastic bonds. The initial regime of continuous elastic deformation of the microtubule is followed by the transition regime, during which the microtubule lattice undergoes discrete structural changes, which include first the reversible dissociation of lateral bonds followed by irreversible dissociation of the longitudinal bonds. We have determined the free energies of dissociation of the lateral (6.9 ± 0.4 kcal/mol) and longitudinal (14.9 ± 1.5 kcal/mol) tubulin–tubulin bonds. These values in conjunction with the large flexural rigidity of tubulin protofilaments obtained (18,000–26,000 pN·nm2) support the idea that the disassembling microtubule is capable of generating a large mechanical force to move chromosomes during cell division. Our computational modeling offers a comprehensive quantitative platform to link molecular tubulin characteristics with the physiological behavior of microtubules. The developed in silico nanoindentation method provides a powerful tool for the exploration of biomechanical properties of other cytoskeletal and multiprotein assemblies.

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

Summary With antibiotic resistance increasing at alarming rates, targets for new antimicrobial therapies must be identified. A particularly promising target is the bacterial two-component system. Two-component systems allow bacteria to detect, evaluate and protect themselves against changes in the environment, such as exposure to antibiotics, and also to trigger production of virulence factors. Drugs that target the response regulator portion of two-component systems represent a potent new approach so far unexploited. Here we focus efforts on the highly virulent bacterium Francisella tularensis tularensis. Francisella contains only three response regulators, making it an ideal system to study. In this study, we initially present the structure of the N-terminal domain of QseB, the response regulator responsible for biofilm formation. Subsequently, using binding assays, computational docking and cellular studies, we show that QseB interacts with 2-aminoimidazole based compounds that impede its function. This information will assist in tailoring compounds to act as adjuvants that will enhance the effect of antibiotics.

Large-size biomolecular systems that spontaneously assemble, disassemble, and self-repair by controlled inputs play fundamental roles in biology. Microtubules (MTs), which play important roles in cell adhesion and cell division, are a prime example. MTs serve as ″tracks″ for molecular motors, and their biomechanical functions depend on dynamic instability-a stochastic switching between periods of rapid growing and shrinking. This process is controlled by many cellular factors so that growth and shrinkage periods are correlated with the life cycle of a cell. Resolving the molecular basis for the action of these factors is of paramount importance for understanding the diverse functions of MTs. We employed a multiscale modeling approach to study the force-induced MT depolymerization by analyzing the mechanical response of a MT protofilament to external forces. We carried out self-organized polymer (SOP) model based simulations accelerated on Graphics Processing Units (GPUs). This approach enabled us to follow the mechanical behavior of the molecule on experimental time scales using experimental force loads. We resolved the structural details and determined the physical parameters that characterize the stretching and bending modes of motion of a MT protofilament. The central result is that the severing action of proteins, such as katanin and kinesin, can be understood in terms of their mechanical coupling to a protofilament. For example, the extraction of tubulin dimers from MT caps by katanin can be achieved by pushing the protofilament toward the axis of the MT cylinder, while the removal of large protofilaments curved into ″ram's horn″ structures by kinesin is the result of the outward bending of the protofilament. We showed that, at the molecular level, these types of deformations are due to the anisotropic, but homogeneous, micromechanical properties of MT protofilaments.

(2015) 52 Tubulin bond energies and microtubule biomechanics determined from nanoindentation insilico, Journal of Biomolecular Structure and Dynamics, 33:sup1,[35][36]