Activation mechanism and novel binding sites of the BKCa channel activator CTIBD.

The large-conductance calcium-activated potassium (BKCa) channel, which is crucial for urinary bladder smooth muscle relaxation, is a potential target for overactive bladder treatment. Our prior work unveiled CTIBD as a promising BKCa channel activator, altering V 1/2 and G max This study investigates CTIBD's activation mechanism, revealing its independence from the Ca2+ and membrane voltage sensing of the BKCa channel. Cryo-electron microscopy disclosed that two CTIBD molecules bind to hydrophobic regions on the extracellular side of the lipid bilayer. Key residues (W22, W203, and F266) are important for CTIBD binding, and their replacement with alanine reduces CTIBD-mediated channel activation. The triple-mutant (W22A/W203A/F266A) channel showed the smallest V 1/2 shift with a minimal impact on activation and deactivation kinetics by CTIBD. At the single-channel level, CTIBD treatment was much less effective at increasing P o in the triple mutant, mainly because of a drastically increased dissociation rate compared with the WT. These findings highlight CTIBD's mechanism, offering crucial insights for developing small-molecule treatments for BKCa-related pathophysiological conditions.

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.

Protonation State of a Bioactive Compound Regulates Its Release from Lamellar Gel-Phase Bilayers.

Lamellar gel networks (LGNs) in personal care or pharmaceutical lotions and creams provide an opaque cream appearance and a creamy texture to these products. Within the LGNs, the lamellar gel (Lß) phase composed of regularly spaced bilayers of surfactants and long-chain fatty alcohols is predominately responsible for the unique rheological properties of the LGNs. To extend the shelf life of LGN-containing products, bioactive compounds with antimicrobial properties are often incorporated into the formulation. However, how the protonation state of the bioactive compounds regulates their release from the Lß-phase bilayers is currently unknown. Using molecular dynamics simulations, we found that the protonated (neutral) form of cinnamic acid, a common antimicrobial food additive, has a retention ratio higher than that of its deprotonated (charged) counterpart in the Lß-phase bilayer. From free energy calculations, we determined that not only is the protonated molecule more stable in the hydrophobic interior of the bilayer but also the formation of hydrogen-bonded dimers significantly enhances its stability within the bilayer. Thus, the protonation state has a profound impact on bioavailability of the compounds. Our results also highlight the importance of considering possible oligomeric states of molecules when performing calculations to estimate the permeability of molecules within various bilayers.

Gramicidin A as a mechanical sensor for mixed nonideal lipid membranes.

Gramicidin A (gA) is a short hydrophobic ß-helical peptide that forms cation-selective channels in lipid membranes in the course of transbilayer dimerization. The length of the gA helix is smaller than the thickness of a typical lipid monolayer. Consequently, elastic deformations of the membrane arise in the configurations of gA monomers, conducting dimer, and the intermediate state of coaxial pair, where gA monomers from opposing membrane monolayers are located one on top of the other. The gA channel is characterized by the average lifetime of the conducting state. The elastic properties of the membrane influence the average lifetime, thus making gA a convenient sensor of membrane elasticity. However, the utilization of gA to investigate the elastic properties of mixed membranes comprising two or more components frequently relies on the assumption of ideality, namely that the elastic parameters of mixed-lipid bilayers depend linearly on the concentrations of the components. Here, we developed a general approach that does not rely on the aforementioned assumption. Instead, we explicitly accounted for the possibility of inhomogeneous lateral distribution of all lipid components, as well as for membrane-mediated lateral interactions of gA monomers, dimer, coaxial pair, and minor lipid components. This approach enabled us to derive unknown elastic parameters of lipid monolayer from experimentally determined lifetimes of gA channel in mixed-lipid bilayers. A general algorithm was formulated that allows the unknown elastic parameters of a lipid monolayer to be obtained using gA as a mechanical sensor.

Fluorescence-Based Proteoliposome Methods to Monitor Redox-Active Transition Metal Transmembrane Translocation by Metal Transporters.

Transmembrane transition metal transporter proteins are central gatekeepers in selectively controlling vectorial metal cargo uptake and extrusion across cellular membranes in all living organisms, thus playing key roles in essential and toxic metal homeostasis. Biochemical characterization of transporter-mediated translocation events and transport kinetics of redox-active metals, such as iron and copper, is challenged by the complexity in generating reconstituted systems in which vectorial metal transport can be studied in real time. We present fluorescence-based proteoliposome methods to monitor redox-active metal transmembrane translocation upon reconstitution of purified metal transporters in artificial lipid bilayers. By encapsulating turn-on/-off iron or copper-dependent sensors in the proteoliposome lumen and conducting real-time transport assays using small unilamellar vesicles (SUVs), in which selected purified Fe(II) and Cu(I) transmembrane importer and exporter proteins have been reconstituted, we provide a platform to monitor metal translocation events across lipid bilayers in real time. The strategy is modular and expandable toward the study of different transporter families featuring diverse metal substrate selectivity and promiscuity.

Evaluation of functional transbilayer coupling in live cells by controlled lipid exchange and imaging fluorescence correlation spectroscopy.

Biophysical coupling between the inner and outer leaflets, known as inter-leaflet or transbilayer coupling, is a fundamental organizational principle in the plasma membranes of live mammalian cells. Lipid-based interactions between the two leaflets are proposed to be a primary mechanism underlying transbilayer coupling. However, there are only a few experimental evidence supporting the existence of such interactions in live cells. This is seemingly due to the lack of experimental strategies to perturb the lipid composition in one leaflet and quantitative techniques to evaluate the biophysical properties of the opposite leaflet. The existing strategies often dependent on immobilization and clustering a component in one of the leaflets and technically demanding biophysical tools to evaluate the effects on the opposing leaflet. In the recent years, the London group developed a simple but elegant method, namely methyl-alpha-cyclodextrin catalyzed lipid exchange (LEX), to efficiently exchange outer leaflet lipids with an exogenous lipid of choice. Here, we adopted this method to perturb outer leaflet lipid composition. The corresponding changes in the inner leaflet is evaluated by comparing the diffusion of lipid probes localized in this leaflet in unperturbed and perturbed conditions. We employed highly multiplexed imaging fluorescence correlation spectroscopy (ImFCS), realized in a commercially available or home-built total internal reflection fluorescence microsocope equipped with a fast and sensitive camera, to determine diffusion coefficient of the lipid probes. Using the combination of LEX and ImFCS, we directly demonstrate lipid-based transbilayer coupling that does not require immobilization of membrane components in live mast cells in resting conditions. Overall, we present a relatively straightforward experimental strategy to evaluate transbilayer coupling quantitively in live cells.

Thickness determination of hydroperoxidized lipid bilayers from medium-resolution cryo-TEM images.

As the primary products of lipid oxidation, lipid hydroperoxides constitute an important class of lipids generated by aerobic metabolism. However, despite several years of effort, the structure of the hydroperoxidized bilayer has not yet been observed under electron microscopy. Here we use a 200 kV Cryo-TEM to image small unilamellar vesicles (SUVs) made (i) of pure POPC or SOPC, (ii) of their pure hydroperoxidized form, and (iii) of their equimolar mixtures. We show that the challenges posed by the determination of the thickness of the hydroperoxidized bilayers under these observation conditions can be addressed by an image analysis method that we developed and describe here.

Applications of phase-separating multi-bilayers in protein-membrane domain interactions.

Synthetic model membranes are important tools to elucidate lipid domain and protein interactions due to predefined lipid compositions and characterizable biophysical properties. Here, we introduce a model membrane with multiple lipid bilayers (multi-bilayers) stacked on a mica substrate that is prepared through a spin-coating technique. The spin-coated multi-bilayers are useful in the study of phase separated membranes with a high cholesterol content, mobile lipids, microscopic and reversible phase separation, and easy conjugation with proteins, which make them a good model to study interactions between proteins and membrane domains.

The effect of hydrostatic pressure on lipid membrane lateral structure.

High pressure is both an environmental challenge to which deep sea biology has to adapt, and a highly sensitive thermodynamic tool that can be used to trigger structural changes in biological molecules and assemblies. Lipid membranes are amongst the most pressure sensitive biological assemblies and pressure can have a large influence on their structure and properties. In this chapter, we will explore the use of high pressure small angle X-ray diffraction and high pressure microscopy to measure and quantify changes in the lateral structure of lipid membranes under both equilibrium high pressure conditions and in response to pressure jumps.

Fluorescence imaging of lamellipodin-mediated biomolecular condensates on solid supported lipid bilayer membranes.

Biomolecular condensates play a major role in numerous cellular processes, including several that occur on the surface of lipid bilayer membranes. There is increasing evidence that cellular membrane trafficking phenomena, including the internalization of the plasma membrane through endocytosis, are mediated by multivalent protein-protein interactions that can lead to phase separation. We have recently found that proteins involved in the clathrin-independent endocytic pathway named Fast Endophilin Mediated Endocytosis can undergo liquid-liquid phase separation (LLPS) in solution and on lipid bilayer membranes. Here, the protein solution concentrations required for phase separation to be observed are significantly smaller compared to those required for phase separation in solution. LLPS is challenging to systematically characterize in cellular systems in general, and on biological membranes in particular. Model membrane approaches are more suitable for this purpose as they allow for precise control over the nature and amount of the components present in a mixture. Here we describe a method that enables the imaging of LLPS domain formation on solid supported lipid bilayers. These allow for facile imaging, provide long-term stability, and avoid clustering of vesicles and vesicle-attached features (such as buds and tethers) in the presence of multi-valent membrane interacting proteins.

Modeling asymmetric cell membranes at all-atom resolution.

Molecular dynamics (MD) simulations are a useful tool when studying the properties of membranes as they allow for a molecular view of lipid interactions with proteins, nucleic acids, or small molecules. While model membranes are usually symmetric in their lipid composition between leaflets and include a small number of lipid components, physiological membranes are highly complex and vary in the level of asymmetry. Simulation studies have shown that changes in leaflet asymmetry can alter the properties of a membrane. It is therefore necessary to carefully build asymmetric membranes to accurately simulate membranes. This chapter carefully describes the different methods for building asymmetric membranes and the advantages/disadvantages of each method. The simplest methods involve building a membrane with either an equal number of lipids per leaflet or an equal initial surface area (SA) estimated by the area per lipid. More detailed methods include combining two symmetric membranes of equal SA or altering an asymmetric membrane and adjusting the number of lipids after equilibration to minimize an observable such as differential stress (0-DS). More complex methods that require specific simulation software are also briefly described. The challenges and assumptions are listed for each method which should help guide the researcher to choose the best method for their unique MD simulation of an asymmetric membrane.

Predicting lipid sorting in curved membranes.

Most biological membranes are curved, and both lipids and proteins play a role in generating curvature. For any given membrane shape and composition, it is not trivial to determine whether lipids are laterally distributed in a homogeneous or inhomogeneous way, and whether the inter-leaflet distribution is symmetric or not. Here we present a simple computational tool that allows to predict the preference of any lipid type for membranes with positive vs. negative curvature, for any given value of curvature. The tool is based on molecular dynamics simulations of tubular membranes with hydrophilic pores. The pores allow spontaneous, barrierless flip-flop of most lipids, while also preventing differences in pressure between the inner and outer water compartments and minimizing membrane asymmetric stresses. Specifically, we provide scripts to build and analyze the simulations. We test the tool by performing simulations on simple binary lipid mixtures, and we show that, as expected, lipids with negative intrinsic curvature distribute to the tubule inner leaflet, the more so when the radius of the tubular membrane is small. Compared to other existing computational methods, relying on membrane buckles and tethers, our method is based on spontaneous inter-leaflet transport of lipids, and therefore allows to explore lipid distribution in asymmetric membranes. The method can easily be adapted to work with any molecular dynamics code and any force field.

The density-threshold affinity: Calculating lipid binding affinities from unbiased coarse-grained molecular dynamics simulations.

Many membrane proteins are sensitive to their local lipid environment. As structural methods for membrane proteins have improved, there is growing evidence of direct, specific binding of lipids to protein surfaces. Unfortunately the workhorse of understanding protein-small molecule interactions, the binding affinity for a given site, is experimentally inaccessible for these systems. Coarse-grained molecular dynamics simulations can be used to bridge this gap, and are relatively straightforward to learn. Such simulations allow users to observe spontaneous binding of lipids to membrane proteins and quantify localized densities of individual lipids or lipid fragments. In this chapter we outline a protocol for extracting binding affinities from these localized distributions, known as the "density threshold affinity." The density threshold affinity uses an adaptive and flexible definition of site occupancy that alleviates the need to distinguish between "bound'' lipids and bulk lipids that are simply diffusing through the site. Furthermore, the method allows "bead-level" resolution that is suitable for the case where lipids share binding sites, and circumvents ambiguities about a relevant reference state. This approach provides a convenient and straightforward method for comparing affinities of a single lipid species for multiple sites, multiple lipids for a single site, and/or a single lipid species modeled using multiple forcefields.

Modeling the mechanochemical feedback for membrane-protein interactions using a continuum mesh model.

The Helfrich free energy is widely used to model the generation of membrane curvature due to different physical and chemical components. The governing equations resulting from the energy minimization procedure are a system of coupled higher order partial differential equations. Simulations of membrane deformation for obtaining quantitative comparisons against experimental observations require computational schemes that will allow us to solve these equations without restrictions to axisymmetric coordinates. Here, we describe one such tool that we developed in our group based on discrete differential geometry to solve these equations along with examples.

Non-affine deformation analysis and 3D packing defects: A new way to probe membrane heterogeneity in molecular simulations.

Here, we discuss a new framework developed over the last 5 years in our group to probe nanoscale membrane heterogeneity. The framework is based on the idea of characterizing lateral heterogeneity through non-affine deformation (NAD) measurements, transverse heterogeneity through three dimensional (3D) lipid packing defects, and using these approaches to formalize the seemingly trivial correlation between lateral organization and lipid packing in biological membranes. We find that measurements from NAD analysis, a prescription which is borrowed from Physics of glasses and granular material, can faithfully distinguish between liquid-ordered and disordered phases in membranes at molecular length scales and, can also be used to identify phase boundaries with high precision. Concomitantly, 3D-packing defects can not only distinguish between the two co-existing fluid phases based on their molecular scale packing (or membrane free volume), but also provide a route to connect the membrane domains to their functionality, such as exploring the molecular origins of inter-leaflet domain registration and peptide partitioning. The correlation between lateral membrane order and transverse packing presents novel molecular design-level features that can explain functions such as protein/peptide partitioning and small-molecule permeation dynamics in complex and heterogeneous membranes with high-fidelity. The framework allows us to explore the nature of lateral organization and molecular packing as a manifestation of intricate molecular interactions among a chemically rich variety of lipids and other molecules in a membrane with complex membrane composition and asymmetry across leaflets.

Analyzing lipid distributions and curvature in molecular dynamics simulations of complex membranes.

We describe methods to analyze lipid distributions and curvature in membranes with complex lipid mixtures and embedded membrane proteins. We discuss issues involved in these analyses, available tools to calculate curvature preferences of lipids and proteins, and focus on tools developed in our group for visual analysis of lipid-protein interactions and the analysis of membrane curvature.

Quantifying uncertainty in trans-membrane stresses and moments in simulation.

The lateral stress profile of a lipid bilayer constitutes a valuable link between molecular simulation and mesoscopic elastic theory. Even though it is frequently calculated in simulations, its statistical precision (or that of observables derived from it) is often left unspecified. This omission can be problematic, as uncertainties are prerequisite to assessing statistical significance. In this chapter, we provide a comprehensive yet accessible overview of the statistical error analysis for the lateral stress profile. We detail two relatively simple but powerful techniques for generating error bars: block-averaging and bootstrapping. Combining these methods allows us to reliably estimate uncertainties, even in the presence of both temporal and spatial correlations, which are ubiquitous in simulation data. We illustrate these techniques with simple examples like stress moments, but also more complex observables such as the location of stress profile extrema and the monolayer neutral surface.

Lattice-based mesoscale simulations and mean-field theory of cell membrane adhesion.

Adhesion of cell membranes involves multi-scale phenomena, ranging from specific molecular binding at Angstrom scale all the way up to membrane deformations and phase separation at micrometer scale. Consequently, theory and simulations of cell membrane adhesion require multi-scale modeling and suitable approximations that capture the essential physics of these phenomena. Here, we present a mesoscale model for membrane adhesion which we have employed in a series of our recent studies. This model quantifies, in particular, how nanoscale lipid clusters physically affect and respond to the intercellular receptor-ligand binding that mediates membrane adhesion. The goal of this Chapter is to present all details and subtleties of the mean-field theory and Monte Carlo simulations of this mesoscale model, which can be used to further explore physical phenomena related to cell membrane adhesion.

The mutual lipid-mediated effect of the transmembrane domain of SARS-CoV-2 E-protein and glycyrrhizin nicotinate derivatives on the localization in the lipid bilayer.

Glycyrrhizinic acid (GA) is one of the active substances in licorice root. It exhibits antiviral activity against various enveloped viruses, for example, SARS-CoV-2. GA derivatives are promising biologically active compounds from perspective of developing broad-spectrum antiviral agents. Given that GA nicotinate derivatives (Glycyvir) demonstrate activity against various DNA- and RNA-viruses, a search for a possible mechanism of action of these compounds is required. In the present paper, the interaction of Glycyvir with the transmembrane domain of the SARS-CoV-2 E-protein (ETM) in a model lipid membrane was investigated by NMR spectroscopy and molecular dynamics simulation. The lipid-mediated influence on localization of the SARS-CoV-2 E-protein by Glycyvir was observed. The presence of Glycyvir leads to deeper immersion of the ETM in lipid bilayer. Taking into account that E-protein plays a significant role in virus production and takes part in virion assembly and budding, the data on the effect of potential antiviral agents on ETM localization and structure in the lipid environment may provide a basis for further studies of potential coronavirus E-protein inhibitors.

Martini 3 Coarse-Grained Model for the Cofactors Involved in Photosynthesis.

As a critical step in advancing the simulation of photosynthetic complexes, we present the Martini 3 coarse-grained (CG) models of key cofactors associated with light harvesting (LHCII) proteins and the photosystem II (PSII) core complex. Our work focuses on the parametrization of beta-carotene, plastoquinone/quinol, violaxanthin, lutein, neoxanthin, chlorophyll A, chlorophyll B, and heme. We derived the CG parameters to match the all-atom reference simulations, while structural and thermodynamic properties of the cofactors were compared to experimental values when available. To further assess the reliability of the parameterization, we tested the behavior of these cofactors within their physiological environments, specifically in a lipid bilayer and bound to photosynthetic complexes. The results demonstrate that our CG models maintain the essential features required for realistic simulations. This work lays the groundwork for detailed simulations of the PSII-LHCII super-complex, providing a robust parameter set for future studies.