Biochimica et Biophysica Acta (BBA) - Biomembranes

Significant cellular processes, including protein sorting, signal transduction, and pathogen entry, amongst others, are associated with membrane microdomains, also known as lipid rafts. Lipid rafts, due to their unique biophysical properties compared to their surrounding environment, which stem from their distinct lipid and protein profiles, have garnered interest in methods and techniques that tune their coexisting liquid-ordered/liquid-disordered state, aiming to disrupt or destabilize them. Since cholesterol stabilizes the membrane domain, cholesterol-depleting compounds like cyclodextrin can be used to destabilize and disrupt the membrane rafts. Overall, given the membrane rafts importance in biological processes, it is crucial to understand the biophysical factors that influence its stability. In this study, we present a new method for disrupting and dissolving lipid rafts in a model system of phase-separated supported lipid bilayer (SLB) patches composed of DOPC, DPPC, and cholesterol. Using fluorescence microscopy to monitor the liquid ordered (Lo) and liquid disordered (Ld) phases of the SLB patches, we observed that adding DOPC liposomes causes a transformation of the co-existing Ld and Lo phases into a single-phase bilayer. On the other hand, adding liposomes that match the lipid content of the phase-separated SLB patch increase the areas of the existing Ld and Lo phases. This work also offers a new method for redistributing raft-localized molecules, confirmed by tracking the redistribution of cholera toxin bound to GM1 after domain dissolution with DOPC liposomes. The work describes an alternative method for dynamically altering membrane composition and dissolving domains via liposome addition, rather than lipid depletion or exchange.

Small-angle X-ray or neutron scattering (SAXS/SANS) analysis of large unilamellar vesicles (LUVs) is often limited by high-dimensional bilayer models and the lack of dedicated, statistically rigorous workflows. Here, we introduce SAS_MoCa, an open-source Python package that integrates a compositional scattering density profile (SDP) description of lipid bilayers with a separated form factor (SFF) treatment of vesicle size and polydispersity, and couples these highly parameterized models to an adaptive thermodynamic simulated annealing algorithm formulated within a constrained Bayesian framework. SAS_MoCa enables users to incorporate quantitative prior information from, e.g., previous SAXS/SANS studies, dynamic light scattering, NMR, or molecular simulations, and returns full posterior parameter distributions, uncertainties (reported as medians and median absolute deviations) and correlations even from single SAXS curves. Validation on POPC, POPE and DMPC SAXS-only data demonstrates that the method yields reproducible structural parameters with uncertainties comparable to joint SAXS/contrast-variation SANS analyses. The modular architecture of SAS_MoCa facilitates extension to additional lipid systems and future joint SAXS/SANS or SANS-only applications.

We present a comparative study of the inner membrane of three Gram-positive bacterial strains, namely S. aureus, S. epidermidis and N. lacusekhoensis. A lipidomics study is used to obtain the lipid architecture and composition for S. epidermidis found in the skin microbiome and N. lacusekhoensis, an extremophile present in halophilic and alkophilic environments. Differences between the strains arise from both the lipid architecture and the cardiolipin content varying from 5% in S. aureus to 85% in N. lacusekhoensis. We develop coarse grained (CG) Martini-3 membrane models which reproduce structural properties such as membrane area, thickness, density distributions as well as ion-correlations with all-atom models. Inter-lipid correlations reveal a homogeneous distribution of lipids in the membranes despite the wide variation in lipid types and composition. Mechanical properties such as the area stretch modulus increased with cardiolipin content, however the bending modulus has a more complex dependence on membrane charge and lipid type. Using the CG models we evaluate the insertion free energies for four widely used antimicrobial molecules. Entry barriers for thymol and methylparaben arise from the charge density modulation at the membrane headgroups due to counterion condensation. The entry mechanisms of the antimicrobial peptide cecropin-melittin-15 (CM15) and the preservative molecule ethyl-lauroyl-arginate (ELAR) are found to be similar across all three strains. We also illustrate the manner in which the extremophilic strain, N. lacusekhoensis with its high cardiolipin content, modulates the partitioning kinetics of the antimicrobial molecule thymol with pH and salt. Our study reveals that membrane properties are largely conserved across the three model membranes. The molecular models and insights emerging from the present work should aid in the development of novel antimicrobials against Gram-positive strains.

Recent genetic and bioinformatic studies have led to the discovery that many bacterial species encode the genes required to produce sphingolipids. Shotgun lipidomic studies have identified numerous sphingolipid species with novel structures that do not exist in eukaryotic organisms. The impacts of these lipids on the biophysical properties of bacterial membranes have not yet been determined. In this study, we purify a novel anionic bacterial sphingolipid, ceramide phosphoglycerate (CPG), and investigate its effect on membrane zeta potential and bending stiffness. CPG and its precursor, ceramide 1-phosphate (C1P), are shown to increase the magnitude of the membrane zeta potential. These sphingolipids also increase the stiffness of these membranes, with CPG increasing rigidity more than C1P or ceramide. This work provides experimental and computational methods of lipid isolation and characterization that may be broadly applicable to a variety of uncharacterized bacterial sphingolipids. SIGNIFICANCEThe diversity of bacterial sphingolipids far exceeds those found in eukaryotes. However, the function and biophysical properties of these lipids are unknown. Characterization of these lipids is a challenge as they are not commercially available. In this study, we developed experimental methods to purify the anionic sphingolipid ceramide phosphoglycerate and incorporate it into liposomes for analysis. Furthermore, we built computational tools to determine the bending stiffness of sphingolipid-containing vesicles from thermal fluctuation data.

The plasma membrane is a laterally heterogeneous environment in which lipid organization plays a central role in regulating protein function. In model systems, this heterogeneity is often described in terms of coexisting liquid-ordered (Lo) and liquid-disordered (Ld) phases, commonly associated with the lipid raft concept. Despite extensive experimental and computational efforts, the molecular determinants governing protein partitioning between these domains remain poorly understood, largely due to the limited number of systems studied. Here, we address this challenge using a high-throughput computational approach, systematically analyzing the partitioning behavior of almost 5,000 helical transmembrane peptides in phase-separating lipid membranes. Across all simulations, we find that none of the peptides exhibit a clear preference for the Lo phase, while the vast majority partition into the Ld phase. This observation is consistent with experimental results in simplified membrane systems and suggests that commonly used ternary lipid mixtures may not fully capture the physicochemical environment governing protein sorting in biological membranes. In addition, we identify a subset of peptides that preferentially localize at the Lo/Ld interface. These interfacial peptides display distinct sequence characteristics, indicating that boundary localization is governed by specific combinations of residue composition and spatial arrangement rather than a single dominant feature. Overall, our results reveal that transmembrane helix partitioning in model membranes is dominated by a preference for disordered environments, with interfacial localization emerging as a distinct and potentially functional behavior.

Lipid droplets (LDs) are cellular organelles responsible for lipid storage and metabolism. The mechanism of biogenesis of LDs involves phase separation of neutral lipids from the surrounding phospholipids, which generates oil lenses embedded in lipid bilayers, also known as nascent LDs. As nascent LDs grow, at some point they bud out of the bilayer, forming nearly spherical droplets. Nascent LDs have different propensity to bud, and it has been proposed that their shape provides information on such propensity; however, LD shape is difficult to determine experimentally. Here we studied the shape of lipid droplets using MD simulations at the coarse-grained level, and compared it to the predictions by an established theory. Our general system setup features an oil lens embedded into a flat, periodic bilayer. We found that the shape of simulated nascent LDs resembles a spherical cap (i.e., it has constant curvature over most of the surface), in excellent agreement with the theory, already for very small droplet sizes. The aspect ratio (height/radius) of nascent LDs increases with increasing LD volume, increasing membrane softness, and increasing surface tension between oil and water, also in agreement with theoretical predictions; however, it remains lower than 1 (i.e., the ratio for a sphere) for LDs of up to 40 nm in diameter. Fitting the simulated LD shapes with a theoretical shape equation suggests that a non-zero surface tension is present in both the monolayer and in the bilayer region. The existence of a relatively high surface tension in the bilayer region is confirmed by local stress calculations, and indicates that the periodic system setup does not reproduce the properties of nascent LDs in the endoplasmic reticulum, where the bilayer tension is two orders of magnitude lower. However, the simulations provide a microscopic view into the properties of droplet embedded vesicles.

Magnetic treatment in tomato seeds (Solanum lycopersicum L.) has been studied as a biotechnological technique to induce a reduction in germination times and enhance plant development. However, the modified cellular mechanisms involved in the reduction of germination times or the improvement of development parameters are not yet clearly established. To explore a possible altered cellular mechanism, the effect of homogeneous static magnetic fields on the structure of the cyclic nucleotide-gated channel 6 (CNGC6), the modification in the organization of POPC lipids in the plasma membrane, and changes in calcium ion mobility were evaluated. For this purpose, coarse-grained molecular dynamics simulations were performed using the Martini 3 model in GROMACS, applying five different magnetic flux densities (0.000, 0.001, 0.010, 0.100, 1.000, and 10.000) T over 1 000 ns. The results showed an anisotropic effect in the longitudinal direction of the protein, which generated heterogeneous behavior among the chains of the homotetramer; this altered the conformation of the CNGC6 channel and modified the pore bottleneck. In contrast, no significant changes were observed in the conformational order of the POPC phospholipid chains. As a preliminary, single-replicate exploratory study, these results suggest that homogeneous static magnetic fields may induce specific structural modifications in the CNGC6 ion channel of Solanum lycopersicum L. without compromising the integrity of the lipid bilayer or the dynamics of ion transport within the analyzed timescale; these preliminary findings provide a molecular-level structural basis for future experimental and computational investigations of magnetic field effects on plant cyclic nucleotide-gated channels.

Integral membrane proteins remodel the surrounding lipid bilayer, but quantifying the resulting deformations and linking them to protein density in the membrane has remained challenging. Here, we introduce an integrative methodology that combines all-atom molecular dynamics (MD) simulations with multiscale small-angle X-ray scattering (SAXS) analysis to connect membrane strain to the protein/lipid ratio in proteoliposomes. Using outer membrane phospholipase A (OmpLA) reconstituted into lipid bilayers with both increased and decreased hydrophobic thickness, we systematically probe the effects of positive and negative hydrophobic mismatch.MD simulations demonstrate that OmpLA causes anisotropic, oscillatory thickness deformations extending up to eight times the radius of the first lipid shell surrounding the protein, yet the net change in average membrane thickness remains below 1%. Through our multiscale SAXS analysis, we quantitatively extract structural parameters, ranging from proteoliposome size to internal membrane architecture, using constrained Bayesian inference, with priors derived from MD findings. Specifically, we determine the protein/lipid molar ratio and average membrane strain, revealing excellent agreement between experiment and simulation. In thinner bilayers, substantial protein loss limits the analysis, highlighting the role of bilayer stability in sample preparation. Moreover, the predominance of OmpLA monomers in the thicker membranes is consistent with weak, membrane-mediated repulsive interactions between protein inclusions. Collectively, this integrative approach establishes a framework for quantifying protein-lipid interactions across molecular and mesoscale dimensions.

Detergent-free extraction of membrane proteins using polymers directly into nanodiscs from the cell membrane has been used widely in recent years. Since the first use of poly(styrene-co-maleic acid) (SMA), numerous related polymers have been developed that differ in chemical architecture and nanodisc characteristics, each capable of influencing the structural and functional properties of the encapsulated membrane protein and its surrounding lipids. Identifying an optimal solubilising polymer, therefore, requires consideration not only of extraction efficiency but also compatibility with downstream applications and analyses. Polymer series in which a single parameter is systematically varied provide a valuable, nuanced tool for optimising nanodisc utility in downstream applications. This study utilises a chemically defined series of poly(styrene-co-maleic acid-co-(N-benzyl)maleimide) (BzAM) terpolymers that exhibit a stepwise, systematic increase in hydrophobicity. Using the human calcitonin gene-related peptide (CGRP) receptor as an exemplar class B1 G-protein-coupled receptor (GPCR), the ability of each BzAM terpolymer to solubilise the receptor from mammalian cell membranes was assessed. All members of the series successfully solubilised CGRP receptor, with solubilisation efficiency correlating positively with increasing hydrophobicity. Importantly, the receptor retained its characteristic high-affinity ligand-binding capability when encapsulated within the BzAM nanodisc, demonstrating that functional integrity is preserved following BzAM-mediated extraction and purification. These findings establish the BzAM terpolymer series as a systematic, tuneable, well-defined tool for the detergent-free solubilisation and functional investigation of GPCRs, and other membrane proteins, in near-native lipid environments. HIGHLIGHTSO_LIStepwise-tuned poly(styrene-co-maleic acid-co-(N-benzyl)maleimide) (BzAM) terpolymers provide a chemically defined, hydrophobicity-controlled platform for detergent-free membrane protein extraction. C_LIO_LIAll BzAM variants effectively solubilise the human calcitonin gene-related peptide (CGRP) receptor, with extraction efficiency increasing in line with terpolymer hydrophobicity. C_LIO_LICGRP receptor maintains high-affinity ligand binding in BzAM nanodiscs, demonstrating preservation of ligand-binding function after solubilisation. C_LIO_LIThe BzAM series provides a novel platform for studying G-protein-coupled receptors and other membrane proteins in near-native lipid environments, with the potential to deliver mechanistic insights and support future drug-discovery efforts. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/726474v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1cb167corg.highwire.dtl.DTLVardef@313e60org.highwire.dtl.DTLVardef@f64a2borg.highwire.dtl.DTLVardef@17f6629_HPS_FORMAT_FIGEXP M_FIG C_FIG

The protozoan parasite Trypanosoma brucei is implicated in deadly African sleeping sickness. Experimental studies show that T. brucei codes for three aquaporin homologs (TbAQP1 to TbAQP3). TbAQP2 has been established as the high affinity drug transporter of drugs pentamidine and melarsoprol. Mutation in TbAQP2 or its loss result in pentamidine-melarsoprol cross-resistance. TbAQP2 is also shown to transport water, glycerol and other solutes to respond to osmoregulation in the infected hosts or glycerol metabolism. Experimentally determined structures of TbAQP2 shows that it adopts the same aquaporin-like hourglass helical fold. However, the so called aromatic/arginine selectivity filter (Ar/R SF) in TbAQP2 has neither arginine nor aromatic residue and all four residues are hydrophobic. Mutation and functional studies have demonstrated the role of Ar/R SF residues in the transport and selectivity of solutes in aquaporin homologs. The intriguing question is how the completely hydrophobic Ar/R SF region enables the transport of water and glycerol molecules. In this study, we used computational approach to elucidate the molecular mechanism of water and glycerol transport. Our equilibrium molecular dynamics simulations showed that the number of water molecules transported by TbAQP2 is almost one order of magnitude higher than that of prototype water channel AQP1. Moreover, the residence time within TbAQP2 channel is much less compared to that found in AQP1. The relatively wider constriction, interactions of water molecules with the selectivity filter residues and the contact duration, all contribute to a large number of water molecules transported through TbAQP2 channel. Our umbrella sampling studies show that when glycerol is transported through TbAQP2, it participates in interactions with channel residues that can be considered as complimentary to that observed in prototype glycerol transporter GlpF. Our studies reveal the molecular mechanism of water and glycerol transport in TbAQP2 and establish that TbAQP2 is an efficient water transporter. Statement of SignificanceTrypanosoma brucei causes African sleeping sickness and a homolog of aquaporin, TbAQP2, is involved in the transport of drugs that are used to treat this disease. Developing anti-parasitic drugs requires the knowledge of molecular mechanism of the proteins function. TbAQP2 has been shown to transport water and glycerol. Permeating solutes have to pass through a narrow constriction region formed by all hydrophobic residues. In the present study, equilibrium molecular dynamics simulations showed that TbAQP2 transports water molecules faster in large quantity in comparison with mammalian AQP1. Higher water transport is due to relatively wider constriction and minimum water interactions with selectivity filter hydrophobic residues. Permeating glycerol molecule is involved in complementary interactions with the channel residues. Our studies reveal how water and glycerol are transported through hydrophobic selectivity filter in TbAQP2.

Saponin-phospholipid bicelles have emerged as promising membrane-mimetic systems for anisotropy-based NMR studies, but their utility depends on their ability to accommodate physiologically relevant lipid compositions and ionic environments. Here, we systematically investigated how saponin chemistry governs three key properties of magnetically alignable bicelles: tolerance to anionic lipid incorporation, responsiveness to lanthanide-induced alignment reorientation, and stability in the presence of divalent metal ions. Using 31P NMR spectroscopy as a sensitive probe of phase behavior and alignment, we compared bicelles formed with glycyrrhizic acid (GA), hederacoside C (HC), and crude Quillaja saponins (CQS). While all saponins effectively solubilized the anionic lipid DMPG, only HC supported magnetically aligned bicelles with high anionic lipid fractions (up to 70%), whereas GA was limited to low incorporation (10%). Both GA- and HC-based bicelles underwent lanthanide-induced alignment flipping, though with significant spectral broadening and intermediate alignment states indicative of increased heterogeneity. Divalent cation effects were strongly ion- and saponin-dependent; HC bicelles were robust to the presence of Ca2+ but were destabilized by Mg2+, while GA bicelles were disrupted by both ions at low concentrations. Together, these results demonstrate that saponin identity critically determines bicelle compatibility with charged lipids and ionic conditions, establishing design principles for tailoring saponin-based bicelles as versatile, biomimetic alignment media for membrane-protein structural studies.

Membrane association is governed by the thermodynamics of amino acid partitioning between water and the lipid bilayer. Here, we quantified amino acid side-chain insertion energetics in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer using unbiased molecular dynamics simulations. Equilibrium depth distributions of 28 analogs, including multiple protonation states, were converted into potentials of mean force (PMFs) by Boltzmann inversion. The resulting PMFs reproduced the main features of bilayer partitioning. Hydrophobic analogs favored the bilayer core, aromatic analogs were stabilized in interfacial regions, and polar or charged analogs remained unfavorable in the hydrophobic interior. A diglycine analog representing the peptide backbone behaved similarly to uncharged polar residues. Depth-dependent pKa profiles and orientational analyses further showed how protonation equilibria and aromatic-ring alignment influence insertion energetics. Agreement with experimental hydrophobicity scales supports the robustness of the approach. These results provide an efficient and internally consistent framework for characterizing bilayer insertion energetics and establish a reference for future studies in more complex lipid environments. O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/723583v1_ufig1.gif" ALT="Figure 1"> View larger version (79K): org.highwire.dtl.DTLVardef@127b12org.highwire.dtl.DTLVardef@14de924org.highwire.dtl.DTLVardef@53b27org.highwire.dtl.DTLVardef@16e8ee4_HPS_FORMAT_FIGEXP M_FIG C_FIG SIGNIFICANCEMembrane-associated proteins represent a large fraction of the proteome and include many major drug targets, yet quantitative understanding of their interactions with lipid bilayers remains limited. Here, we present an unbiased molecular dynamics framework for systematically determining amino acid side-chain insertion free energies in a model bilayer. By deriving potentials of mean force directly from equilibrium depth distributions, this approach enables internally consistent comparisons across residue classes and protonation states without biasing restraints. The resulting free-energy profiles reproduce established hydrophobicity trends and show how protonation equilibria and aromatic-ring orientation modulate bilayer partitioning. This scalable strategy provides a quantitative reference for residue-level membrane thermodynamics and establishes a foundation for extending insertion energetics to more diverse lipid compositions and more complex membrane-associated systems.

Plasma membrane sheets generated by controlled mechanical disruption provide direct access to the cytosolic face of the plasma membrane while preserving the native organization of membrane-associated proteins and lipids. Here, we present a protocol for generating and validating sonication-derived plasma membrane sheets from cultured cells, primary neurons, and isolated synaptosomes. We further describe their application for live and fixed imaging of membrane protein localization, organization, conformational dynamics, and protein-protein interactions, as well as quantitative membrane-associated synaptic vesicle recruitment assays. This versatile platform preserves the native membrane environment while enabling direct visualization and quantitative analysis of membrane-associated processes at high spatial resolution. The protocol can be readily adapted to investigate diverse membrane proteins, lipid-dependent mechanisms, and vesicle tethering events across a wide range of cellular systems.

TAT (Twin-Arginine Translocation) is a preprotein translocation system dedicated to membrane translocation of prefolded proteins in plants and bacteria. The TAT translocon, consisting of the TatABC subunits, drives translocation using proton motive force. However, there have been no reports on the successful reconstitution of the TAT system. In this report, we show that MPIase, a glycolipid that has known to catalyze membrane protein integration, is essential for the TAT system. Our findings in recombinant Escherichia coli demonstrate that overproducing TatABC increases MPIase levels and that depleting MPIase results in TAT precursor accumulation in the cytosol. Furthermore, co-reconstitution of MPIase with TatABC revealed the translocation activities of TAT substrates in a proton motive force-dependent manner. This is the first successful reconstitution of the TAT system and will be advantageous for understanding its mechanisms.

Images in the electron microscope are formed by electron scattering and focusing. The spherical geometry of these processes gives rise to two coherent, conjugate spherical wave fronts, known as Ewald spheres. These spheres are associated with the two halves of the contrast transfer function (CTF), and their widths are determined by the focal gradient through the specimen. To properly correct for the CTF, each half of the CTF must be applied to an image individually and integrated into the reconstruction into the corresponding Ewald sphere. Theory indicates that this dual Ewald sphere reconstruction method should recover the maximal amount of information possible. This method was compared to the other reconstruction methods commonly used: the projection approximation (ignoring the Ewald sphere), the simple insertion and the single sideband methods. In simulated reconstructions the dual Ewald sphere method recovered the most information when the correct half of the CTF is matched to the corresponding Ewald sphere. If the wrong half is matched, the result worse than the projection approximation method. Examining reconstructions from real data indicated that the dual Ewald sphere method performs at least as well as the simple insertion method, but not as good as in simulations. The likely reason is the two-fold ambiguity in the assigned orientations of the particle images, which remains an issue to pursue in further studies. In conclusion, the dual Ewald sphere reconstruction method may offer the best way to calculate very high resolution reconstructions when the micrograph quality warrants it. HighlightsO_LIThe dual Ewald sphere reconstruction corrects for the two halves of the CTF. C_LIO_LIThe signs of the two halves of the CTF must correspond to the focal gradient. C_LIO_LIDetermining the focal gradient for individual particle images remains unresolved. C_LIO_LIComplex reconstructions indicate any real space phases are artifacts. C_LI

A proteomic analysis of Ixodes scapularis nymph saliva identified 252 proteins, including six tubular lipid-binding proteins (TULIPs). Comparing nymphs fed on mice that were uninfected or infected with Borrelia burgdorferi, twelve salivary proteins showed significant differences in the amounts detected, including XP_040079658.2, which we refer to as TULIP2. Considering the known immunity-related functions of some TULIPs, we expressed and purified TULIP2 from Escherichia coli and analyzed its interaction with B. burgdorferi lipids. The purification of TULIP2 from E. coli presented many obstacles, due to insolubility, which is consistent with previous reports from studies of other TULIP family members. The binding results showed specificity for B. burgdorferi lipids, with evidence for cholesteryl {beta}-galactoside as a major binding target. Molecular modeling of TULIP2 did not show any strong lipid binding sites. We used molecular dynamics simulation of TULIP2 to explore its conformational landscape by thermal unfolding. The earliest unfolding intermediate opened a hydrophobic pocket to which cholesteryl {beta}-galactoside was predicted to bind strongly. We propose that a specific lipid bilayer interaction with TULIP2 triggers the opening of the ligand-binding site.

The ability to assign amino acid resonances in multidimensional NMR spectra of biomolecules is necessary for detailed studies of protein structure and dynamics. Despite creative advances in isotopic labeling, unlabeling and multidimensional NMR experiments, resonance assignment remains a bottleneck in studies of large proteins. In this work, we show that the metabolic flux through biosynthetic pathways of amino acid production during protein expression can be modulated to aid in the identification of resonances in two-dimensional NMR spectra. This straightforward method involves doping 15N-enriched minimal media with small amounts of rich natural abundance media to generate unique peak intensity attenuation patterns, producing type-specific signatures of amino acids in two-dimensional 15N HSQC experiments. Using three model proteins, IGPS (51 kDa heterodimer), PTP1B (35 kDa), PHPT1 (14 kDa), we show that this method can disentangle several amino acid types, is robust to different expression conditions, and is a useful supplement for triple resonance experiments in protein backbone resonance assignments.

Production of recombinant proteins frequently yields inclusion bodies that must undergo refolding to yield active protein. Here, we optimized the refolding conditions for the recombinant leucyl aminopeptidase (rPepL) from Lactocaseibacillus casei expressed in inclusion bodies from E. coli. Several chemical additives were assessed for how well they facilitated an increase in refolding efficiency. The best, 0.5 M L-arginine, yielded 50.8% refolding. The addition of stabilizers, such as sucrose and glycerol, with L-arginine further increased yields to 85%. Urea at lower concentrations (0.25-0.5 M) also facilitated an increase in the refolding yield when co-added with L-arginine, whereas guanidinium chloride inhibited it. Sugars and polyols exhibited dose-dependent effects, with ranges for optima also defined. Fluorescence spectroscopy verified enhancements in the refolding under the optimized conditions. Molecular dynamics simulation under mixed solvent conditions provided atomic insights about stabilizing interactions that are likely to facilitate increased refolding. The results show that a series of aggregation suppressors and protein stabilizers can, in a collaborative way, increase the refolding efficiency for the recombinant proteins from the inclusion bodies. The protocol with the optimization using the additives L-arginine, sucrose, and glycerol is an efficient method for the production of active rPepL. This article outlines the best refolding method to recover recombinant leucyl aminopeptidase from inclusion bodies of E. coli using L-arginine combined with sucrose and glycerol. The combined experimental observations and computational simulations elucidate the molecular process of additive-induced stabilization, which elucidates how aggregation inhibition and hydrogen-bonded stabilization act synergistically. The results presented herein answer both mechanistic understanding and experimental guidance for improving protein refolding.

Staphyloxanthin (STX) is a carotenoid synthesized by the human pathogen Staphylococcus aureus. The golden color of this bacterium is due to this carotenoid. STX protects Staphylococcus aureus from oxidative stress by scavenging free radical species. Furthermore, STX has been shown to mechanically strengthen the Staphylococcus aureus membrane and to form microdomains that recruit antibiotic-resistance factors. Thus, inhibition of STX is a promising strategy for intervening against multidrug-resistant strains of this pathogen. However, the molecular mechanisms by which STX regulates the membrane structure and function of Staphylococcus aureus remain unclear. More specifically, the localization of STX within phosphatidylglycerol (PG) bilayers, the primary phospholipid of this bacteriums membrane, and how this localization drives macroscopic biophysical changes remain unresolved questions. Here, we addressed this issue by integrating molecular dynamics (MD) simulations, X-ray scattering experiments, and fluorescence spectroscopy. We developed an atomistic model of STX, which was validated against X-ray scattering data and which is suitable for all-atom MD simulations. We demonstrate that STX significantly increases lipid packing and acyl chain order of STX-PG bilayer mixtures. In addition, STX self-assembles into clusters, where the long and rigid conjugated triterpenoid chain interdigitates across both leaflets, modifying locally the density of the surrounding PG molecules. These findings provide a molecular explanation to the reduced headgroup spacing and core dynamics observed in fluorescence experiments and are consistent with the formation of structurally-distinct STX-enriched microdomains. Notably, STX reduces the gel-to-liquid crystalline phase transition temperature, indicating a general stabilizing effect for the fluid phase of PG lipids of varying length. Overall, our findings provide molecular insights into how STX enhances membrane mechanical integrity. It will be highly interesting to establish how the membrane remodeling effects observed here connect with STXs dual roles, acting as an antioxidant and preventing pore formation and other mechanical perturbations induced by antimicrobial molecules.

Bordetella produce a wide array of virulence factors. These factors are involved in bacterial colonization and evasion of immune defenses. Our recent studies revealed that the bacteria produce an exoglycan, Bordetella oligosaccharide (BOS). B. petrii is the evolutionary early divergent species of the genus Bordetella. This study has focused on the investigation of two B. petrii type strains: clinical and environmental. We employed nuclear magnetic resonance (NMR) analyses to elucidate the structural differences between their lipopolysaccharides. Our findings revealed that the LPS of clinical B. petrii strain comprises a hexasaccharide unit, that was structurally identical to the BOS. This form of LPS is only a minor population in the bacterial outer membrane of the environmental strain. In addition to the cell-bound BOS, its secreted glycoform was also found in growth media of B. petrii. Anti-BOS neoglycoconjugate antibodies cross-reacted with B. petrii LPS. This suggest that the newly identified BOS associated with B. petrii PS would be a potential vaccine element against Bordetella.