Thicket and Mesh: How the Outer Membrane Can Resist Tension Imposed by the Cell Wall.

The cell envelope of Gram-negative bacteria is composed of an outer membrane (OM) and an inner membrane (IM) and a peptidoglycan cell wall (CW) between them. Combined with Braun's lipoprotein (Lpp), which connects the OM and the CW, and numerous membrane proteins that exist in both OM and IM, the cell envelope creates a mechanically stable environment that resists various physical and chemical perturbations to the cell, including turgor pressure caused by the solute concentration difference between the cytoplasm of the cell and the extracellular environment. Previous computational studies have explored how individual components (OM, IM, and CW) can resist turgor pressure although combinations of them have been less well studied. To that end, we constructed multiple OM-CW systems, including the Lpp connections with the CW under increasing degrees of strain. The results show that the OM can effectively resist the tension imposed by the CW, shrinking by only 3-5% in area even when the CW is stretched to 2.5× its relaxed area. The area expansion modulus of the system increases with increasing CW strain, although the OM remains a significant contributor to the envelope's mechanical stability. Additionally, we find that when the protein TolC is embedded in the OM, its stiffness increases.

Extraction of Dielectric Permittivity from Atomistic Molecular Dynamics Simulations and Microwave Measurements.

The design of new biotechnology depends on the prediction and measurement of the electrical properties of biomolecules. The dielectric permittivity, in particular, is highly important for the design of microwave systems for diagnostics, yet this property is rarely explicitly targeted during the development of biomolecular force fields for molecular dynamics (MD) simulations. In order to explore the ability of existing force fields to reproduce the frequency-dependent permittivity, we carried out MD simulations of various aqueous solutions, including pure water, isopropyl alcohol, alanine, and the protein ubiquitin. The TIP3P, TIP4P, TIP4P/ε, and SWM4-NDP water models were used along with the CHARMM36m and Drude protein force fields. An experimental setup using a truncated coaxial line was created to measure the permittivity of the same solutions to check for measure-model agreement. We found that one of the nonpolarizable force fields (TIP4P/ε + CHARMM36m) and the polarizable force fields (SWM4-NDP + Drude) closely agree with experimental results. This demonstrates the strength of the tuned TIP4P/ε water model, as well as the physical validity of polarizable force fields in capturing dielectric permittivity. This represents an important step toward the predictive design of biosensors.

The Role of Extracellular Loops in the Folding of Outer Membrane Protein X (OmpX) of Escherichia coli.

The outer membrane of Gram-negative bacteria acts as an additional diffusion barrier for solutes and nutrients. It is perforated by outer membrane proteins (OMPs) that function most often as diffusion pores, but sometimes also as parts of larger cellular transport complexes, structural components of the cell wall, or even as enzymes. These OMPs often have large loops that protrude into the extracellular environment, which have promise for biotechnological applications and as therapeutic targets. Thus, understanding how modifications to these loops affect OMP stability and folding is critical for their efficient application. In this work, the small outer membrane protein OmpX was used as a model system to quantify the effects of loop insertions on OMP folding and stability. The insertions were varied according to both hydrophobicity and size, and their effects were determined by assaying folding into detergent micelles in vitro by SDS-PAGE and in vivo by isolating the outer membrane of cells expressing the constructs. The different insertions were also examined in molecular dynamics simulations to resolve how they affect OmpX dynamics in its native outer membrane. The results indicate that folding of OMPs is affected by both the insert length and by its hydrophobic character. Small insertions sometimes even improved the folding efficiency of OmpX, while large hydrophilic inserts reduced it. All the constructs that were found to fold in vitro could also do so in their native environment. One construct that could not fold in vitro was transported to the OM in vivo, but remained unfolded. Our results will help to improve the design and efficiency of recombinant OMPs used for surface display.

Modeling intermediates of BamA folding an outer membrane protein.

BamA, the core component of the ß-barrel assembly machinery complex, is an integral outer-membrane protein (OMP) in Gram-negative bacteria that catalyzes the folding and insertion of OMPs. A key feature of BamA relevant to its function is a lateral gate between its first and last ß-strands. Opening of this lateral gate is one of the first steps in the asymmetric-hybrid-barrel model of BamA function. In this study, multiple hybrid-barrel folding intermediates of BamA and a substrate OMP, EspP, were constructed and simulated to better understand the model's physical consequences. The hybrid-barrel intermediates consisted of the BamA ß-barrel and its POTRA5 domain and either one, two, three, four, five, or six ß-hairpins of EspP. The simulation results support an asymmetric-hybrid-barrel model in which the BamA N-terminal ß-strand forms stronger interactions with the substrate OMP than the C-terminal ß-strand. A consistent "B"-shaped conformation of the final folding intermediate was observed, and the shape of the substrate ß-barrel within the hybrid matched the shape of the fully folded substrate. Upon further investigation, inward-facing glycines were found at sharp bends within the hybrid and fully folded ß-barrels. Together, the data suggest an influence of sequence on shape of the substrate barrel throughout the OMP folding process and of the fully folded OMP.

Adaptation of the periplasm to maintain spatial constraints essential for cell envelope processes and cell viability.

The cell envelope of Gram-negative bacteria consists of two membranes surrounding a periplasm and peptidoglycan layer. Molecular machines spanning the cell envelope depend on spatial constraints and load-bearing forces across the cell envelope and surface. The mechanisms dictating spatial constraints across the cell envelope remain incompletely defined. In Escherichia coli, the coiled-coil lipoprotein Lpp contributes the only covalent linkage between the outer membrane and the underlying peptidoglycan layer. Using proteomics, molecular dynamics, and a synthetic lethal screen, we show that lengthening Lpp to the upper limit does not change the spatial constraint but is accommodated by other factors which thereby become essential for viability. Our findings demonstrate E. coli expressing elongated Lpp does not simply enlarge the periplasm in response, but the bacteria accommodate by a combination of tilting Lpp and reducing the amount of the covalent bridge. By genetic screening, we identified all of the genes in E. coli that become essential in order to enact this adaptation, and by quantitative proteomics discovered that very few proteins need to be up- or down-regulated in steady-state levels in order to accommodate the longer Lpp. We observed increased levels of factors determining cell stiffness, a decrease in membrane integrity, an increased membrane vesiculation and a dependance on otherwise non-essential tethers to maintain lipid transport and peptidoglycan biosynthesis. Further this has implications for understanding how spatial constraint across the envelope controls processes such as flagellum-driven motility, cellular signaling, and protein translocation.

Plasticity within the barrel domain of BamA mediates a hybrid-barrel mechanism by BAM.

In Gram-negative bacteria, the biogenesis of ß-barrel outer membrane proteins is mediated by the ß-barrel assembly machinery (BAM). The mechanism employed by BAM is complex and so far- incompletely understood. Here, we report the structures of BAM in nanodiscs, prepared using polar lipids and native membranes, where we observe an outward-open state. Mutations in the barrel domain of BamA reveal that plasticity in BAM is essential, particularly along the lateral seam of the barrel domain, which is further supported by molecular dynamics simulations that show conformational dynamics in BAM are modulated by the accessory proteins. We also report the structure of BAM in complex with EspP, which reveals an early folding intermediate where EspP threads from the underside of BAM and incorporates into the barrel domain of BamA, supporting a hybrid-barrel budding mechanism in which the substrate is folded into the membrane sequentially rather than as a single unit.

Inward-facing glycine residues create sharp turns in ß-barrel membrane proteins.

The transmembrane region of outer-membrane proteins (OMPs) of Gram-negative bacteria are almost exclusively ß-barrels composed of between 8 and 26 ß-strands. To explore the relationship between ß-barrel size and shape, we modeled and simulated engineered variants of the Escherichia coli protein OmpX with 8, 10, 12, 14, and 16 ß-strands. We found that while smaller barrels maintained a roughly circular shape, the 16-stranded variant developed a flattened cross section. This flat cross section impeded its ability to conduct ions, in agreement with previous experimental observations. Flattening was determined to arise from the presence of inward-facing glycines at sharp turns in the ß-barrel. An analysis of all simulations revealed that glycines, on average, make significantly smaller angles with residues on neighboring strands than all other amino acids, including alanine, and create sharp turns in ß-barrel cross sections. This observation was generalized to 119 unique structurally resolved OMPs. We also found that the fraction of glycines in ß-barrels decreases as the strand number increases, suggesting an evolutionary role for the addition or removal of glycine in OMP sequences.

BamA is required for autotransporter secretion.

BACKGROUND: In Gram-negative bacteria, type Va and Vc autotransporters are proteins that contain both a secreted virulence factor (the "passenger" domain) and a ß-barrel that aids its export. While it is known that the folding and insertion of the ß-barrel domain utilize the ß-barrel assembly machinery (BAM) complex, how the passenger domain is secreted and folded across the membrane remains to be determined. The hairpin model states that passenger domain secretion occurs independently through the fully-formed and membrane-inserted ß-barrel domain via a hairpin folding intermediate. In contrast, the BamA-assisted model states that the passenger domain is secreted through a hybrid of BamA, the essential subunit of the BAM complex, and the ß-barrel domain of the autotransporter. METHODS: To ascertain the models' plausibility, we have used molecular dynamics to simulate passenger domain secretion for two autotransporters, EspP and YadA. RESULTS: We observed that each protein's ß-barrel is unable to accommodate the secreting passenger domain in a hairpin configuration without major structural distortions. Additionally, the force required for secretion through EspP's ß-barrel is more than that through the BamA ß-barrel. CONCLUSIONS: Secretion of autotransporters most likely occurs through an incompletely formed ß-barrel domain of the autotransporter in conjunction with BamA. GENERAL SIGNIFICANCE: Secretion of virulence factors is a process used by practically all pathogenic Gram-negative bacteria. Understanding this process is a necessary step towards limiting their infectious capacity.

Detection and Quantification of Hydrogen Peroxide in Aqueous Solutions Using Chemical Exchange Saturation Transfer.

The development of new analytical methods to accurately quantify hydrogen peroxide is of great interest. In the current study, we developed a new magnetic resonance (MR) method for noninvasively quantifying hydrogen peroxide (H2O2) in aqueous solutions based on chemical exchange saturation transfer (CEST), an emerging MRI contrast mechanism. Our method can detect H2O2 by its specific CEST signal at ∼6.2 ppm downfield from water resonance, with more than 1000 times signal amplification compared to the direct NMR detection. To improve the accuracy of quantification, we comprehensively investigated the effects of sample properties on CEST detection, including pH, temperature, and relaxation times. To accelerate the NMR measurement, we implemented an ultrafast Z-spectroscopic (UFZ) CEST method to boost the acquisition speed to 2 s per CEST spectrum. To accurately quantify H2O2 in unknown samples, we also implemented a standard addition method, which eliminated the need for predetermined calibration curves. Our results clearly demonstrate that the presented CEST-based technique is a simple, noninvasive, quick, and accurate method for quantifying H2O2 in aqueous solutions.