Dynamic Second Harmonic Imaging of Proton Translocation Through Water Needles in Lipid Membranes.

Proton translocation through lipid membranes is a fundamental process in the field of biology. Several theoretical models have been developed and presented over the years to explain the phenomenon, yet the exact mechanism is still not well understood. Here, we show that proton translocation is directly related to membrane potential fluctuations. Using high-throughput wide-field second harmonic (SH) microscopy, we report apparently universal transmembrane potential fluctuations in lipid membrane systems. Molecular simulations and free energy calculations suggest that H+ permeation proceeds predominantly across a thin, membrane-spanning water needle and that the transient transmembrane potential drives H+ ions across the water needle. This mechanism differs from the transport of other cations that require completely open pores for transport and follows naturally from the well-known Grotthuss mechanism for proton transport in bulk water. Furthermore, SH imaging and conductivity measurements reveal that the rate of proton transport depends on the structure of the hydrophobic core of bilayer membranes.

Hairpin protein partitioning from the ER to lipid droplets involves major structural rearrangements.

Lipid droplet (LD) function relies on proteins partitioning between the endoplasmic reticulum (ER) phospholipid bilayer and the LD monolayer membrane to control cellular adaptation to metabolic changes. It has been proposed that these hairpin proteins integrate into both membranes in a similar monotopic topology, enabling their passive lateral diffusion during LD emergence at the ER. Here, we combine biochemical solvent-accessibility assays, electron paramagnetic resonance spectroscopy and intra-molecular crosslinking experiments with molecular dynamics simulations, and determine distinct intramembrane positionings of the ER/LD protein UBXD8 in ER bilayer and LD monolayer membranes. UBXD8 is deeply inserted into the ER bilayer with a V-shaped topology and adopts an open-shallow conformation in the LD monolayer. Major structural rearrangements are required to enable ER-to-LD partitioning. Free energy calculations suggest that such structural transition is unlikely spontaneous, indicating that ER-to-LD protein partitioning relies on more complex mechanisms than anticipated and providing regulatory means for this trans-organelle protein trafficking.

Passive transport of Ca2+ ions through lipid bilayers imaged by widefield second harmonic microscopy.

In biology, release of Ca2+ ions in the cytosol is essential to trigger or control many cell functions. Calcium signaling acutely depends on lipid membrane permeability to Ca2+. For proper understanding of membrane permeability to Ca2+, both membrane hydration and the structure of the hydrophobic core must be taken into account. Here, we vary the hydrophobic core of bilayer membranes and observe different types of behavior in high-throughput wide-field second harmonic imaging. Ca2+ translocation is observed through mono-unsaturated (DOPC:DOPA) membranes, reduced upon the addition of cholesterol, and completely inhibited for branched (DPhPC:DPhPA) and poly-unsaturated (SLPC:SLPA) lipid membranes. We propose, using molecular dynamics simulations, that ion transport occurs through ion-induced transient pores, which requires nonequilibrium membrane restructuring. This results in different rates at different locations and suggests that the hydrophobic structure of lipids plays a much more sophisticated regulating role than previously thought.

Ion-Induced Transient Potential Fluctuations Facilitate Pore Formation and Cation Transport through Lipid Membranes.

Unassisted ion transport through lipid membranes plays a crucial role in many cell functions without which life would not be possible, yet the precise mechanism behind the process remains unknown due to its molecular complexity. Here, we demonstrate a direct link between membrane potential fluctuations and divalent ion transport. High-throughput wide-field non-resonant second harmonic (SH) microscopy of membrane water shows that membrane potential fluctuations are universally found in lipid bilayer systems. Molecular dynamics simulations reveal that such variations in membrane potential reduce the free energy cost of transient pore formation and increase the ion flux across an open pore. These transient pores can act as conduits for ion transport, which we SH image for a series of divalent cations (Cu2+, Ca2+, Ba2+, Mg2+) passing through giant unilamellar vesicle (GUV) membranes. Combining the experimental and computational results, we show that permeation through pores formed via an ion-induced electrostatic field is a viable mechanism for unassisted ion transport.

Is Lipid Specificity Key to the Potential Antiviral Activity of Mouthwash Reagent Chlorhexidine against SARS-CoV-2?

Chlorhexidine (CHX), a popular antibacterial drug, is widely used for oral health. Emerging pieces of evidence suggest that commercially available chlorhexidine mouthwash formulations are effective in suppressing the spread of SARS-CoV-2, possibly through destabilization of the viral lipid envelope. CHX is known for its membrane-active properties; however, the molecular mechanism revealing how it damages the viral lipid envelope is yet to be understood. Here we used extensive conventional and umbrella sampling simulations to quantify the effects of CHX on model membranes mimicking the composition of the SARS-CoV-2 outer lipid membrane as well as the host plasma membrane. Our results show that the lipid composition and physical properties of the membrane play an important role in binding and insertion, with CHX binding favorably to the viral membrane over the plasma membrane. Among the simulated lipids, CHX preferentially binds to anionic lipids, PS and PI, which are more concentrated in the viral membrane. The deeper and stable binding of CHX to the viral membrane results in more pronounced swelling of the membrane laterally with a thinning of the bilayer. The overall free energies of pore formation are strongly reduced for the viral membrane compared to the plasma membrane; however, CHX has a larger concentration-dependent effect on free energies of pore formation in the plasma membrane than the viral membrane. The results indicate that CHX is less toxic to the human plasma membrane at low concentrations. Our simulations reveal that CHX facilitates pore formation by the combination of thinning the membrane and accumulation at the water defect. This study provides insights into the mechanism underlying the anti-SARS-CoV-2 potency of CHX, supporting its potential for application as an effective and safe oral rinse agent for preventing viral transmission.

Lipid Droplets Embedded in a Model Cell Membrane Create a Phospholipid Diffusion Barrier.

Lipid droplets (LDs) are ubiquitous, cytoplasmic fat storage organelles that originate from the endoplasmic reticulum (ER) membrane. They are composed of a core of neutral lipids surrounded by a phospholipid monolayer. Proteins embedded into this monolayer membrane adopt a monotopic topology and are crucial for regulated lipid storage and consumption. A key question is, which collective properties of protein-intrinsic and lipid-mediated features determine spatio-temporal protein partitioning between phospholipid bilayer and LD monolayer membranes. To address this question, a freestanding phospholipid bilayer with physiological lipidic composition is produced using microfluidics and micrometer-sized LDs are dispersed around the bilayer that spontaneously insert into the bilayer. Using confocal microscopy, the 3D geometry of the reconstituted LDs is determined with high spatial resolution. The micrometer-sized bilayer-embedded LDs present a characteristic lens shape that obeys predictions from equilibrium wetting theory. Fluorescence recovery after photobleaching measurements reveals the existence of a phospholipid diffusion barrier at the monolayer-bilayer interface. Coarse-grained molecular dynamics simulation reveals lipid specific density distributions along the pore rim, which may rationalize the diffusion barrier. The lipid diffusion barrier between the LD covering monolayer and the bilayer may be a key phenomenon influencing protein partitioning between the ER membrane and LDs in living cells.

Free energies of membrane stalk formation from a lipidomics perspective.

Many biological membranes are asymmetric and exhibit complex lipid composition, comprising hundreds of distinct chemical species. Identifying the biological function and advantage of this complexity is a central goal of membrane biology. Here, we study how membrane complexity controls the energetics of the first steps of membrane fusions, that is, the formation of a stalk. We first present a computationally efficient method for simulating thermodynamically reversible pathways of stalk formation at coarse-grained resolution. The method reveals that the inner leaflet of a typical plasma membrane is far more fusogenic than the outer leaflet, which is likely an adaptation to evolutionary pressure. To rationalize these findings by the distinct lipid compositions, we computed ~200 free energies of stalk formation in membranes with different lipid head groups, tail lengths, tail unsaturations, and sterol content. In summary, the simulations reveal a drastic influence of the lipid composition on stalk formation and a comprehensive fusogenicity map of many biologically relevant lipid classes.

Cooperative Effects of an Antifungal Moiety and DMSO on Pore Formation over Lipid Membranes Revealed by Free Energy Calculations.

Itraconazole is a triazole drug widely used in the treatment of fungal infections, and it is in clinical trials for treatment of several cancers. However, the drug suffers from poor solubility, while experiments have shown that itraconazole delivery in liposome nanocarriers improves both circulation half-life and tissue distribution. The drug release mechanism from the nanocarrier is still unknown, and it depends on several factors including membrane stability against defect formation. In this work, we used molecular dynamics simulations and potential of mean force (PMF) calculations to quantify the influence of itraconazole on pore formation over lipid membranes, and we compared the effect by itraconazole with a pore-stabilizing effect by the organic solvent dimethyl sulfoxide (DMSO). According to the PMFs, both itraconazole and DMSO greatly reduce the free energy of pore formation, by up to ∼20 kJ mol-1. However, whereas large concentrations of itraconazole of 8 mol % (relative to lipid) were required, only small concentrations of a few mole % DMSO (relative to water) were sufficient to stabilize pores. In addition, itraconazole and DMSO facilitate pore formation by different mechanisms. Whereas itraconazole predominantly aids the formation of a partial defect with a locally thinned membrane, DMSO mainly stabilizes a transmembrane water needle by shielding it from the hydrophobic core. Notably, the two distinct mechanisms act cooperatively upon adding both itraconazole and DMSO to the membrane, as revealed by an additional reduction of the pore free energy. Overall, our simulations reveal molecular mechanisms and free energies of membrane pore formation by small molecules. We suggest that the stabilization of a locally thinned membrane as well as the shielding of a transmembrane water needle from the hydrophobic membrane core may be a general mechanism by which amphiphilic molecules facilitate pore formation over lipid membranes at sufficient concentrations.

Complexity of seemingly simple lipid nanodiscs.

Lipid nanodiscs are macromolecular assemblies, where a scaffold protein is wrapped around a nanosized disc of a lipid bilayer, thus protecting the hydrocarbon chains at the disc edges from unfavorable interactions with water. These nanostructures have numerous applications in, e.g., nanotechnology and pharmaceutics, and in investigations of membrane proteins. Here, we present results based on atomistic molecular dynamics simulations combined with electron paramagnetic spectroscopy measurements on the structure and dynamics of lipids in single-component nanodiscs. Our data highlight the existence of three distinctly different lipid fractions: central lipids residing in the center of a nanodisc, boundary lipids in direct contact with a scaffold protein, and intermediate lipids between these two regions. The central lipids are highly ordered and characterized by slow diffusion. In this part of the nanodisc, the membrane is the thickest and characterized by a gel-like or liquid-ordered phase, having features common to cholesterol-rich membranes. The boundary lipids in direct contact with the scaffold protein turned out to be less ordered and characterized by faster diffusion, and they remained in the liquid-disordered phase even at temperatures that were somewhat below the main phase transition temperature (Tm). The enthalpies associated with the central-boundary and central-intermediate transitions were similar to those observed for lipids going through the main phase transition. Overall, the study reveals lipid nanodiscs to be characterized by a complex internal structure, which is expected to influence membrane proteins placed in nanodiscs.

Computer simulations of protein-membrane systems.

The interactions between proteins and membranes play critical roles in signal transduction, cell motility, and transport, and they are involved in many types of diseases. Molecular dynamics (MD) simulations have greatly contributed to our understanding of protein-membrane interactions, promoted by a dramatic development of MD-related software, increasingly accurate force fields, and available computer power. In this chapter, we present available methods for studying protein-membrane systems with MD simulations, including an overview about the various all-atom and coarse-grained force fields for lipids, and useful software for membrane simulation setup and analysis. A large set of case studies is discussed.

Cholesterol Reduces Partitioning of Antifungal Drug Itraconazole into Lipid Bilayers.

Cholesterol plays a crucial role in modulating the physicochemical properties of biomembranes, both increasing mechanical strength and decreasing permeability. Cholesterol is also a common component of vesicle-based delivery systems, including liposome-based drug delivery systems (LDSs). However, its effect on the partitioning of drug molecules to lipid membranes is very poorly recognized. Herein, we performed a combined experimental/computational study of the potential for the use of the LDS formulation for the delivery of the antifungal drug itraconazole (ITZ). We consider the addition of cholesterol to the lipid membrane. Since ITZ is only weakly soluble in water, its bioavailability is limited. Use of an LDS has thus been proposed. We studied lipid membranes composed of cholesterol, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), and ITZ using a combination of computational molecular dynamics (MD) simulations of lipid bilayers and Brewster angle microscopy (BAM) experiments of monolayers. Both experimental and computational results show separation of cholesterol and ITZ. Cholesterol has a strong preference to orient parallel to the bilayer normal. However, ITZ, a long and relatively rigid molecule with weakly hydrophilic groups along the backbone, predominantly locates below the interface between the hydrocarbon chain region and the polar region of the membrane, with its backbone oriented parallel to the membrane surface; the orthogonal orientation in the membrane could be the cause of the observed separation. In addition, fluorescence measurements demonstrated that the affinity of ITZ for the lipid membrane is decreased by the presence of cholesterol, which is thus probably not a suitable formulation component of an LDS designed for ITZ delivery.

Mechanism of homodimeric cytokine receptor activation and dysregulation by oncogenic mutations.

Homodimeric class I cytokine receptors are assumed to exist as preformed dimers that are activated by ligand-induced conformational changes. We quantified the dimerization of three prototypic class I cytokine receptors in the plasma membrane of living cells by single-molecule fluorescence microscopy. Spatial and spatiotemporal correlation of individual receptor subunits showed ligand-induced dimerization and revealed that the associated Janus kinase 2 (JAK2) dimerizes through its pseudokinase domain. Oncogenic receptor and hyperactive JAK2 mutants promoted ligand-independent dimerization, highlighting the formation of receptor dimers as the switch responsible for signal activation. Atomistic modeling and molecular dynamics simulations based on a detailed energetic analysis of the interactions involved in dimerization yielded a mechanistic blueprint for homodimeric class I cytokine receptor activation and its dysregulation by individual mutations.

Role of Oxidized Gly25, Gly29, and Gly33 Residues on the Interactions of Aß1-42 with Lipid Membranes.

Oxidative stress is known to play an important role in the pathogenesis of Alzheimer's disease. Moreover, it is becoming increasingly evident that the plasma membrane of neurons plays a role in modulating the aggregation and toxicity of Alzheimer's amyloid-ß peptide (Aß). In this study, the combined and interdependent effects of oxidation and membrane interactions on the 42 residues long Aß isoform are investigated using molecular simulations. Hamiltonian replica exchange molecular dynamics simulations are utilized to elucidate the impact of selected oxidized glycine residues of Aß42 on the interactions of the peptide with a model membrane comprised of 70% POPC, 25% cholesterol, and 5% of the ganglioside GM1. The main findings are that, independent of the oxidation state, Aß prefers binding to GM1 over POPC, which is further enhanced by the oxidation of Gly29 and Gly33 and reduced the formation of ß-sheet. Our results suggest that the differences observed in Aß42 conformations and its interaction with a lipid bilayer upon oxidation originate from the position of the oxidized Gly residue with respect to the hydrophobic sequence of Aß42 involving the Gly29-XXX-Gly33-XXX-Gly37 motif and from specific interactions between the peptide and the terminal sugar groups of GM1.

Cryo-EM structure of the complete and ligand-saturated insulin receptor ectodomain.

Glucose homeostasis and growth essentially depend on the hormone insulin engaging its receptor. Despite biochemical and structural advances, a fundamental contradiction has persisted in the current understanding of insulin ligand-receptor interactions. While biochemistry predicts two distinct insulin binding sites, 1 and 2, recent structural analyses have resolved only site 1. Using a combined approach of cryo-EM and atomistic molecular dynamics simulation, we present the structure of the entire dimeric insulin receptor ectodomain saturated with four insulin molecules. Complementing the previously described insulin-site 1 interaction, we present the first view of insulin bound to the discrete insulin receptor site 2. Insulin binding stabilizes the receptor ectodomain in a T-shaped conformation wherein the membrane-proximal domains converge and contact each other. These findings expand the current models of insulin binding to its receptor and of its regulation. In summary, we provide the structural basis for a comprehensive description of ligand-receptor interactions that ultimately will inform new approaches to structure-based drug design.

Cholesteryl Hemisuccinate Is Not a Good Replacement for Cholesterol in Lipid Nanodiscs.

Nanodiscs are suitable tools for studies of membrane proteins (MPs) due to their ability to mimic native biological membranes, and several MP structures are solved in nanodiscs. Among the various cell membrane components, cholesterol (CHL) is known to regulate protein function and its concentration can reach up to 50 mol %. However, studies comprising cholesterol are challenging due to its hydrophobic nature, hence, nanodiscs with only a low cholesterol concentration have been studied. To overcome the problem, cholesterol analogs with high solubility in polar solutions are often used, and one of them is cholesteryl hemisuccinate (CHS). Nevertheless, in molecular dynamics (MD) simulation, this is not an obstacle. In this study, we performed MD simulations of nanodiscs containing neutral phosphatidylcholine (POPC) lipids, negatively charged phosphatidylglycerol (POPG) lipids, CHL, or negatively charged cholesterol analog, CHS. Our simulations show that CHS increases the order of lipids in nanodiscs; the effect is, however, weaker than CHL and even smaller in nanodiscs. Furthermore, CHS gathered around scaffold proteins while cholesterol was uniformly distributed in the nanodiscs. Thus, nanodiscs with CHS are heterogeneous and not equivalent to nanodiscs with CHL. Finally, we also observed the increased concentration of POPG near the scaffold proteins, driven by electrostatic interactions. The MD results are experimentally validated using electron paramagnetic resonance spectroscopy. These results show that nanodiscs are, in fact, complex structures not easily comparable with planar lipid bilayers.

Behavior of the DPH fluorescence probe in membranes perturbed by drugs.

1,6-Diphenyl-1,3,5-hexatriene (DPH) is one of the most commonly used fluorescent probes to study dynamical and structural properties of lipid bilayers and cellular membranes via measuring steady-state or time-resolved fluorescence anisotropy. In this study, we present a limitation in the use of DPH to predict the order of lipid acyl chains when the lipid bilayer is doped with itraconazole (ITZ), an antifungal drug. Our steady-state fluorescence anisotropy measurements showed a significant decrease in fluorescence anisotropy of DPH embedded in the ITZ-containing membrane, suggesting a substantial increase in membrane fluidity, which indirectly indicates a decrease in the order of the hydrocarbon chains. This result or its interpretation is in disagreement with the fluorescence recovery after photobleaching measurements and molecular dynamics (MD) simulation data. The results of these experiments and calculations indicate an increase in the hydrocarbon chain order. The MD simulations of the bilayer containing both ITZ and DPH provide explanations for these observations. Apparently, in the presence of the drug, the DPH molecules are pushed deeper into the hydrophobic membrane core below the lipid double bonds, and the probe predominately adopts the orientation of the ITZ molecules that is parallel to the membrane surface, instead of orienting parallel to the lipid acyl chains. For this reason, DPH anisotropy provides information related to the less ordered central region of the membrane rather than reporting the properties of the upper segments of the lipid acyl chains.

Structural insights from lipid-bilayer nanodiscs link α-Synuclein membrane-binding modes to amyloid fibril formation.

The protein α-Synuclein (αS) is linked to Parkinson's disease through its abnormal aggregation, which is thought to involve cytosolic and membrane-bound forms of αS. Following previous studies using micelles and vesicles, we present a comprehensive study of αS interaction with phospholipid bilayer nanodiscs. Using a combination of NMR-spectroscopic, biophysical, and computational methods, we structurally and kinetically characterize αS interaction with different membrane discs in a quantitative and site-resolved way. We obtain global and residue-specific αS membrane affinities, and determine modulations of αS membrane binding due to αS acetylation, membrane plasticity, lipid charge density, and accessible membrane surface area, as well as the consequences of the different binding modes for αS amyloid fibril formation. Our results establish a structural and kinetic link between the observed dissimilar binding modes and either aggregation-inhibiting properties, largely unperturbed aggregation, or accelerated aggregation due to membrane-assisted fibril nucleation.

Physiologically-relevant levels of sphingomyelin, but not GM1, induces a ß-sheet-rich structure in the amyloid-ß(1-42) monomer.

To resolve the contribution of ceramide-containing lipids to the aggregation of the amyloid-ß protein into ß-sheet rich toxic oligomers, we employed molecular dynamics simulations to study the effect of cholesterol-containing bilayers comprised of POPC (70% POPC, and 30% cholesterol) and physiologically relevant concentrations of sphingomyelin (SM) (30% SM, 40% POPC, and 30% cholesterol), and the GM1 ganglioside (5% GM1, 70% POPC, and 25% cholesterol). The increased bilayer rigidity provided by SM (and to a lesser degree, GM1) reduced the interactions between the SM-enriched bilayer and the N-terminus of Aß42 (and also residues Ser26, Asn27, and Lys28), which facilitated the formation of a ß-sheet in the normally disordered N-terminal region. Aß42 remained anchored to the SM-enriched bilayer through hydrogen bonds with the side chain of Arg5. With ß-sheets in the at the N and C termini, the structure of Aß42 in the sphingomyelin-enriched bilayer most resembles ß-sheet-rich structures found in higher-ordered Aß fibrils. Conversely, when bound to a bilayer comprised of 5% GM1, the conformation remained similar to that observed in the absence of GM1, with Aß42 only making contact with one or two GM1 molecules. This article is part of a Special Issue entitled: Protein Aggregation and Misfolding at the Cell Membrane Interface edited by Ayyalusamy Ramamoorthy.

doGlycans-Tools for Preparing Carbohydrate Structures for Atomistic Simulations of Glycoproteins, Glycolipids, and Carbohydrate Polymers for GROMACS.

Carbohydrates constitute a structurally and functionally diverse group of biological molecules and macromolecules. In cells they are involved in, e.g., energy storage, signaling, and cell-cell recognition. All of these phenomena take place in atomistic scales, thus atomistic simulation would be the method of choice to explore how carbohydrates function. However, the progress in the field is limited by the lack of appropriate tools for preparing carbohydrate structures and related topology files for the simulation models. Here we present tools that fill this gap. Applications where the tools discussed in this paper are particularly useful include, among others, the preparation of structures for glycolipids, nanocellulose, and glycans linked to glycoproteins. The molecular structures and simulation files generated by the tools are compatible with GROMACS.

Key steps in unconventional secretion of fibroblast growth factor 2 reconstituted with purified components.

FGF2 is secreted from cells by an unconventional secretory pathway. This process is mediated by direct translocation across the plasma membrane. Here, we define the minimal molecular machinery required for FGF2 membrane translocation in a fully reconstituted inside-out vesicle system. FGF2 membrane translocation is thermodynamically driven by PI(4,5)P2-induced membrane insertion of FGF2 oligomers. The latter serve as dynamic translocation intermediates of FGF2 with a subunit number in the range of 8-12 FGF2 molecules. Vectorial translocation of FGF2 across the membrane is governed by sequential and mutually exclusive interactions with PI(4,5)P2 and heparan sulfates on opposing sides of the membrane. Based on atomistic molecular dynamics simulations, we propose a mechanism that drives PI(4,5)P2 dependent oligomerization of FGF2. Our combined findings establish a novel type of self-sustained protein translocation across membranes revealing the molecular basis of the unconventional secretory pathway of FGF2.