The time step of atomistic molecular dynamics (MD) simulations is determined by the fastest motions in the system and is typically limited to 2 fs. An increasingly popular approach is to increase the mass of the hydrogen atoms to ∼3 amu and decrease the mass of the parent atom by an equivalent amount. This approach, known as hydrogen-mass repartitioning (HMR), permits time steps up to 4 fs with reasonable simulation stability. While HMR has been applied in many published studies to date, it has not been extensively tested for membrane-containing systems. Here, we compare the results of simulations of a variety of membranes and membrane–protein systems run using a 2 fs time step and a 4 fs time step with HMR. For pure membrane systems, we find almost no difference in structural properties, such as area-per-lipid, electron density profiles, and order parameters, although there are differences in kinetic properties such as the diffusion constant. Conductance through a porin in an applied field, partitioning of a small peptide, hydrogen-bond dynamics, and membrane mixing show very little dependence on HMR and the time step. We also tested a 9 Å cutoff as compared to the standard CHARMM cutoff of 12 Å, finding significant deviations in many properties tested. We conclude that HMR is a valid approach for membrane systems, but a 9 Å cutoff is not.

The cell envelope in Gram-negative bacteria comprises two distinct membranes with a cell wall between them. There has been a growing interest in the mechanical adaptation of this cell envelope to the osmotic pressure (or turgor pressure), which is generated by the difference in the concentration of solutes between the cytoplasm and the external environment. However, it remains unexplored how the cell wall, the inner membrane (IM), and the outer membrane (OM) effectively protect the cell from this pressure by bearing the resulting surface tension, thus preventing the formation of inner membrane bulges, abnormal cell morphology, spheroplasts and cell lysis. In this study, we have used molecular dynamics (MD) simulations combined with experiments to resolve how and to what extent models of the IM, OM, and cell wall respond to changes in surface tension. We calculated the area compressibility modulus of all three components in simulations from tension-area isotherms. Experiments on monolayers mimicking individual leaflets of the IM and OM were also used to characterize their compressibility. While the membranes become softer as they expand, the cell wall exhibits significant strain stiffening at moderate to high tensions. We integrate these results into a model of the cell envelope in which the OM and cell wall share the tension at low turgor pressure (0.3 atm) but the tension in the cell wall dominates at high values (> 1 atm).

Objectives This work measured the amount of bound versus unbound water in completely-demineralized dentin. Methods Dentin beams prepared from extracted human teeth were completely demineralized, rinsed and dried to constant mass. They were rehydrated in 41% relative humidity (RH), while gravimetrically measuring their mass increase until the first plateau was reached at 0.064 (vacuum) or 0.116 g H2O/g dry mass (Drierite). The specimens were then exposed to 60% RH until attaining the second plateau at 0.220 (vacuum) or 0.191 g H2O/g dry mass (Drierite), and subsequently exposed to 99% RH until attaining the third plateau at 0.493 (vacuum) or 0.401 g H2O/g dry mass (Drierite). Results Exposure of the first layer of bound water to 0% RH for 5 min produced a −0.3% loss of bound water; in the second layer of bound water it caused a −3.3% loss of bound water; in the third layer it caused a −6% loss of bound water. Immersion in 100% ethanol or acetone for 5 min produced a 2.8 and 1.9% loss of bound water from the first layer, respectively; it caused a −4 and −7% loss of bound water in the second layer, respectively; and a −17 and −23% loss of bound water in the third layer.. Bound water represented 21–25% of total dentin water. Chemical dehydration of water-saturated dentin with ethanol/acetone for 1 min only removed between 25 to 35% of unbound water, respectively. Significance Attempts to remove bound water by evaporation were not very successful. Chemical dehydration with 100% acetone was more successful than 100% ethanol especially the third layer of bound water. Since unbound water represents between 75–79% of total matrix water, the more such water can be removed, the more resin can be infiltrated.

Gram-negative bacteria have a cell envelope that comprises an outer membrane (OM), a peptidoglycan (PG) layer and an inner membrane (IM) 1 . The OM and PG are load-bearing, selectively permeable structures that are stabilized by cooperative interactions between IM and OM proteins 2 , 3 . In E. coli , Braun’s lipoprotein (Lpp) forms the only covalent tether between the OM and PG and is crucial for cell envelope stability 4 but most other Gram-negative bacteria lack Lpp so it has been assumed that alternative mechanisms of OM stabilization are present 5 . We use a glycoproteomic analysis of PG to show that β-barrel OM proteins are covalently attached to PG in several Gram-negative species, including Coxiella burnetii , Agrobacterium tumefaciens and Legionella pneumophila . In C. burnetii , we found that four different types of covalent attachments occur between OM proteins and PG, with tethering of the β-barrel OM protein BbpA becoming most abundant in stationary phase and tethering of the lipoprotein LimB similar throughout the cell-cycle. Using a genetic approach, we demonstrate that the cell-cycle dependent tethering of BbpA is partly dependent on a developmentally regulated L,D transpeptidase (Ldt). We use our findings to propose a model of Gram-negative cell envelope stabilization that includes cell-cycle control and an expanded role for Ldts in covalently attaching surface proteins to PG.

Lipopolysaccharide (LPS) O-antigen (O-Ag) is known to limit antibody binding to surface antigens, although the relationship between antibody, O-Ag and other outer-membrane antigens is poorly understood. Here we report, immunization with the trimeric porin OmpD from Salmonella Typhimurium (STmOmpD) protects against infection. Atomistic molecular dynamics simulations indicate this is because OmpD trimers generate footprints within the O-Ag layer sufficiently sized for a single IgG Fab to access. While STmOmpD differs from its orthologue in S. Enteritidis (SEn) by a single amino-acid residue, immunization with STmOmpD confers minimal protection to SEn. This is due to the OmpD-O-Ag interplay restricting IgG binding, with the pairing of OmpD with its native O-Ag being essential for optimal protection after immunization. Thus, both the chemical and physical structure of O-Ag are key for the presentation of specific epitopes within proteinaceous surface-antigens. This enhances combinatorial antigenic diversity in Gram-negative bacteria, while reducing associated fitness costs.