The intrinsic load-resisting capacity of kinesin

Conventional kinesin is a homodimeric motor protein that is capable of walking unidirectionally along a cytoskeletal filament. While previous experiments indicated unyielding unidirectionality against an opposing load up to the so-called stall force, recent experiments also observed limited processive backwalking under superstall loads. This theoretical study seeks to elucidate the molecular mechanical basis for kinesin's steps over the full range of external loads that can possibly be applied to the dimer. We found that kinesin's load-resisting capacity is largely determined by a synergic ratchet-and-pawl mechanism inherent in the dimer. Load susceptibility of this inner molecular mechanical mechanism underlies kinesin's response to various levels of external loads. Computational implementation of the mechanism enabled us to rationalize major trends observed experimentally in kinesin's stalemate and consecutive back steps. The study also predicts several distinct features of kinesin's load-affected motility, which are seemingly counterintuitive but readily verifiable by future experiment.Comment: 44 pages, 6 figure

Engineering DNA polymerases for application in DNB-based sequencing technology

DNA polymerases serve as the core engine to afford sequence information in sequencing technologies that have revolutionized modern biological research. For application in the DNB-based sequencing platform, an assemblage of DNA polymerases was engineered to catalyze the requisite biochemical reaction. In the process, naturally occurring polymerases were tapped into through deep-learning algorithms for constraints between individual protein residues to narrow down the protein sequence space and to annotate protein sequences in light of their catalytic properties. And the constraints were subsequently applied in designing potential polymerase candidates with the guidance of the sequence annotations. Additionally, ancestral protein sequences were estimated to expand the candidate repertoire. Furthermore, the candidates were subjected to in silico screening before examined by an HTS methodology based on fluorescence signal. Finally, the resulting proteins were expressed and purified for testing in the DNB-based sequencing platform. Our sequencing data suggested that these proteins behave better than their existing counterparts

Simulation on mechanical and failure characteristics of sandstone with elliptical hole under tension-shear effect

Under the influence of geological environment and engineering disturbance, elliptical hole-defects exist widely in engineering rock mass. Excavation unloading causes rebound tensile stress in rock mass. The tension-shear stress zone is formed under the hole defects, which induces the tension-shear failure of rock mass and greatly reduce the stability of engineering rock mass. In order to study the mechanical properties and failure behavior of the rock mass with an elliptical hole under tension and shear, the numerical model was built using discrete element numerical simulation based on the rock mechanics test results. Furthermore, the tension-shear numerical modelling tests of rock mass with an elliptical hole of different hole inclination angle α and the ratio of long to short axis k were carried out, and the meso-mechanism of crack evolution was revealed from the point of view of stress tensor. The results show that when k is constant, with the increase of α, the shear strength approximately shows a “W” shape under low normal tensile stress (1–3 MPa), and the minimum value is obtained when α is 120° or 150°, and the maximum value is obtained when α is 90°. Under high normal tensile stress (4–6 MPa), the shear strength increases at first and then decreases, and the minimum and maximum values are obtained when α is 0° and 90°, respectively. When α is constant, for the rock mass with an elliptical hole if α is not 90°, the shear strength decreases nonlinearly with the increase of k. The sensitivity of stress concentration of the elliptical hole to normal tensile stress decreases at first and then increases with the increase of α, and the sensitivity is the highest when α is 0°. The sensitivity is the lowest when α is 90°, and the sensitivity is higher when α is 120°and 150° than that when α is 30°and 60°. The strength of the rock mass with an elliptical hole is obviously worse than that of intact rock mass, and the degree of deterioration is positively related to the normal tensile stress. The level of crack initiation stress increases with the increase of normal tensile stress, and the crack initiation angle decreases with the increase of normal tensile stress. The failure type of the rock mass with an elliptical hole under tension and shear is the tensile failure caused by anti-wing crack penetration. Under the effect of tension and shear, the maximum tensile zone is formed by the coupling of tensile stress and compressive stress in the rock mass, and the boundary near the side of the shear loading surface is the crack propagation path. The crack starts from the plastic yield at the elliptical hole. After the crack initiation, the stress is released and redistributed by the particle contact fracture, and the crack propagates along the direction of the maximum principal stress after the redistribution, which shows the nonlinear propagation mode of the crack macroscopically

TLR5 signaling enhances the proliferation of human allogeneic CD40-activated B cell induced CD4hiCD25+ regulatory T cells

Although diverse functions of different toll-like receptors (TLR) on human natural regulatory T cells have been demonstrated recently, the role of TLR-related signals on human induced regulatory T cells remain elusive. Previously our group developed an ex vivo high-efficient system in generating human alloantigen-specific CD4(hi)CD25(+) regulatory T cells from naive CD4(+)CD25(-) T cells using allogeneic CD40-activated B cells as stimulators. In this study, we investigated the role of TLR5-related signals on the generation and function of these novel CD4(hi)CD25(+) regulatory T cells. It was found that induced CD4(hi)CD25(+) regulatory T cells expressed an up-regulated level of TLR5 compared to their precursors. The blockade of TLR5 using anti-TLR5 antibodies during the co-culture decreased CD4(hi)CD25(+) regulatory T cells proliferation by induction of S phase arrest. The S phase arrest was associated with reduced ERK1/2 phosphorylation. However, TLR5 blockade did not decrease the CTLA-4, GITR and FOXP3 expressions, and the suppressive function of CD4(hi)CD25(+) regulatory T cells. In conclusion, we discovered a novel function of TLR5-related signaling in enhancing the proliferation of CD4(hi)CD25(+) regulatory T cells by promoting S phase progress but not involved in the suppressive function of human CD40-activated B cell-induced CD4(hi)CD25(+) regulatory T cells, suggesting a novel role of TLR5-related signals in the generation of induced regulatory T cells.published_or_final_versio

The aminobisphosphonate pamidronate controls influenza pathogenesis by expanding a γδ T cell population in humanized mice

As shown in humanized mice, a population of Vγ9Vδ2 T cells can reduce the severity and mortality of disease caused by infection with human and avian influenza viruses

Empirical Optimization of Interactions between Proteins and Chemical Denaturants in Molecular Simulations

Chemical denaturants are the most commonly used perturbation applied to study protein stability and folding kinetics as well as the properties of unfolded polypeptides. We build on recent work balancing the interactions of proteins and water, and accurate models for the solution properties of urea and guanidinium chloride, to develop a combined force field that is able to capture the strength of interactions between proteins and denaturants. We use solubility data for a model tetraglycine peptide in each denaturant to tune the protein-denaturant interaction by a novel simulation methodology. We validate the results against data for more complex sequences: single-molecule Förster resonance energy transfer data for a 34-residue fragment of the globular protein CspTm and photoinduced electron transfer quenching data for the disordered peptides C(AGQ)nW in denaturant solution as well as the chemical denaturation of the mini-protein Trp cage. The combined force field model should aid our understanding of denaturation mechanisms and the interpretation of experiment

Kinesin Is an Evolutionarily Fine-Tuned Molecular Ratchet-and-Pawl Device of Decisively Locked Direction

Conventional kinesin is a dimeric motor protein that transports membranous organelles toward the plus-end of microtubules (MTs). Individual kinesin dimers show steadfast directionality and hundreds of consecutive steps, yetthe detailed physical mechanism remains unclear. Here we compute free energies for the entire dimer-MT system for all possible interacting configurations by taking full account of molecular details. Employing merely first principles and several measured binding and barrier energies, the system-level analysis reveals insurmountable energy gaps between configurations, asymmetric ground state caused by mechanically lifted configurational degeneracy, and forbidden transitions ensuring coordination between both motor domains for alternating catalysis. This wealth of physical effects converts a kinesin dimer into a molecular ratchet-and-pawl device, which determinedly locks the dimer's movement into the MT plus-end and ensures consecutive steps in hand-over-hand gait.Under a certain range of extreme loads, however, the ratchet-and-pawl device becomes defective but not entirely abolished to allow consecutive back-steps. This study yielded quantitative evidence that kinesin's multiple molecular properties have been evolutionarily adapted to fine-tune the ratchet-and-pawl device so as to ensure the motor's distinguished performance.Comment: 10 printed page