Biology subjects

Doktorova, M.

Publications and source records attributed to Doktorova, M..

11 recordsLinked to original sources

bioRxiv2026

Structural basis of caveolin-driven membrane bending

Caveolins are monotopic membrane proteins essential for caveolae formation and have key roles in signaling and lipid regulation. Caveolins assemble into amphipathic discs with a central {beta}-barrel, an architecture distinct from other membrane-remodeling proteins. These discs embed in the membrane inducing membrane curvature. However, the mechanism of disc-driven bending remains unresolved. Using cryo-electron tomography, structure-guided mutagenesis, and mammalian cell studies, we show that evolutionarily distinct caveolins differ dramatically in their ability to curve membranes despite their conserved architecture. Through computational and theoretical analyses, we demonstrate that patterning of hydrophobic residues along the rim of the disc of human Caveolin-1 induces the deformation of the surrounding leaflet, dictating membrane bending. Finally, we determine a 4.1[A] resolution structure of Caveolin-1 within heterologous caveolae in situ, showing the disc adopts a funnel-like conformation, further shaping membrane architecture. Together, these findings reveal fundamental structural principles that empower caveolins to sculpt and remodel cellular membranes.

cell biology↗

bioRxiv2024

QUANTIFYING ACYL CHAIN INTERDIGITATION IN SIMULATED BILAYERS VIA DIRECT TRANSBILAYER INTERACTIONS

In a lipid bilayer, the interactions between lipid hydrocarbon chains from opposing leaflets can influence membrane properties. These interactions include the phenomenon of interdigitation, in which an acyl chain of one leaflet extends past the bilayer midplane and into the opposing leaflet. While static interdigitation is well understood in gel phase bilayers from X-ray diffraction measurements, much less is known about dynamic interdigitation in fluid phases. In this regard, atomistic molecular dynamics simulations can provide mechanistic information on interleaflet interactions that can be used to generate experimentally testable hypotheses. To address limitations of existing computational methodologies which provide results that are either indirect or averaged over time and space, here we introduce three novel ways of quantifying the extent of chain interdigitation. Our protocols include the analysis of instantaneous interactions at the level of individual carbon atoms, thus providing temporal and spatial resolution for a more nuanced picture of dynamic interdigitation. We compare the methods on bilayers composed of lipids with equal total number of carbon atoms but different mismatches between the sn-1 and sn-2 chain lengths. We find that these metrics, which are based on freely available software packages and are easy to implement, provide complementary details that help characterize various features of lipid-lipid contacts at the bilayer midplane. The new frameworks thus allow for a deeper look at fundamental molecular mechanisms underlying bilayer structure and dynamics, and present a valuable expansion of the membrane biophysics toolkit.

biophysics↗

bioRxiv2024

Caveolin assemblies displace one bilayer leaflet to organize and bend membranes

Caveolin is a monotopic integral membrane protein, widely expressed in metazoa and responsible for constructing enigmatic membrane invaginations known as caveolae. Recently, the high-resolution structure of a purified human caveolin assembly, the CAV1-8S complex, revealed a unique organization of 11 protomers arranged in a tightly packed, radially symmetric spiral disc. One face and the outer rim of this disc are hydrophobic, suggesting that the complex incorporates into membranes by displacing hundreds of lipids from one leaflet. The feasibility of this unique molecular architecture and its biophysical and functional consequences are currently unknown. Using Langmuir film balance measurements, we find that CAV1-8S is highly surface active, intercalating into lipid monolayers of various compositions. CAV1-8S can also incorporate into preformed bilayers, but only upon removal of phospholipids from the outer-facing leaflet. Atomistic and coarse-grained simulations of biomimetic bilayers support this leaflet replacement model and also reveal that CAV1-8S accumulates 40-70 cholesterol molecules into a disordered monolayer between the complex and its distal lipid leaflet. We find that CAV1-8S preferentially associates with positively curved membrane surfaces due to its influence on the conformations of distal leaflet lipids, and that these effects laterally sort lipids. Large-scale simulations of multiple caveolin assemblies confirmed their association with large, positively curved membrane morphologies consistent with the shape of caveolae. Further, association with curved membranes regulates the exposure of caveolin residues implicated in protein-protein interactions. Altogether, the unique structure of CAV1-8S imparts unusual modes of membrane interaction with implications for membrane organization, morphology, and physiology. STATEMENT OF SIGNIFICANCECaveolae are membrane invaginations heavily implicated in cellular physiology and disease; however, how their unique shape and function are produced remains enigmatic. Here, following on recent characterization of the unusual structure of the CAV1-8S oligomer, we examine the molecular details of its interactions with its surrounding lipid membrane using simulations and reconstitution experiments. We describe a novel mode of membrane interaction-which we term leaflet replacement-for the CAV1-8S complex that has not previously been observed for any other protein. The biophysical consequences of this unique molecular organization provide mechanistic insights into the functions and organization of caveolae in cells.

biophysics↗

bioRxiv2024

Cryo-EM images of phase separated lipid bilayer vesicles analyzed with a machine learning approach

Lateral lipid heterogeneity (i.e., raft formation) in biomembranes plays a functional role in living cells. Three-component mixtures of low- and high-melting lipids plus cholesterol offer a simplified experimental model for raft domains in which a liquid-disordered (Ld) phase coexists with a liquid-ordered (Lo) phase. Using such models, we recently showed that cryogenic electron microscopy (cryo-EM) can detect phase separation in lipid vesicles based on differences in bilayer thickness. However, the considerable noise within cryo-EM data poses a significant challenge for accurately determining the membrane phase state at high spatial resolution. To this end, we have developed an image processing pipeline that utilizes machine learning (ML) to predict the bilayer phase in projection images of lipid vesicles. Importantly, the ML method exploits differences in both the thickness and molecular density of Lo compared to Ld, which leads to improved phase identification. To assess accuracy, we used artificial images of phase-separated lipid vesicles generated from all-atom molecular dynamics simulations of Lo and Ld phases. Synthetic ground truth datasets mimicking a series of compositions along a tieline of Ld+Lo coexistence were created and then analyzed with various ML models. For all tieline compositions, we find that the ML approach can correctly identify the bilayer phase at 5 nm lateral resolution with > 90% accuracy, thus providing a means to isolate the intensity profiles of coexisting Ld and Lo phases, as well as accurately determine domain size distributions, number of domains, and phase area fractions. The method described here provides a framework for characterizing nanoscopic lateral heterogeneities in membranes and paves the way for a more detailed understanding of raft properties in biological contexts. SignificanceLipid rafts are important for cell function, but in most cases cannot be detected with conventional optical microscopy because of their extremely small size. Cryogenic electron microscopy (cryo-EM), because of its much greater spatial resolution, is capable of imaging domains as small as 5-10 nm. In this report, we show how machine learning techniques can be used to automatically and accurately identify raft-like domains in simulated cryo-EM images, a powerful approach that could ultimately lead to a better understanding of raft properties.

biophysics↗

bioRxiv2023

Cell Membranes Sustain Phospholipid Imbalance Via Cholesterol Asymmetry

Membranes are molecular interfaces that compartmentalize cells to control the flow of nutrients and information. These functions are facilitated by diverse collections of lipids, nearly all of which are distributed asymmetrically between the two bilayer leaflets. Most models of biomembrane structure and function often include the implicit assumption that these leaflets have similar abundances of phospholipids. Here, we show that this assumption is generally invalid and investigate the consequences of lipid abundance imbalances in mammalian plasma membranes (PM). Using quantitative lipidomics, we discovered that cytoplasmic leaflets of human erythrocyte membranes have >50% overabundance of phospholipids compared to exoplasmic leaflets. This imbalance is enabled by an asymmetric interleaflet distribution of cholesterol, which regulates cellular cholesterol homeostasis. These features produce unique functional characteristics, including low PM permeability and resting tension in the cytoplasmic leaflet that regulates protein localization. These largely overlooked aspects of membrane asymmetry represent an evolution of classic paradigms of biomembrane structure and physiology.

biophysics↗

bioRxiv2023

Sampling Bottleneck in Validating Membrane Dynamics

Molecular dynamics simulations of lipid membranes have become increasingly impactful in biophysics because they offer atomistic resolution of structural fluctuations in relation to their functional outputs. Yet quantitative characterization of multiscale processes is a formidable challenge due to the distribution of motions that evade analysis of discrete simulation data. Here we investigate the efficient calculation of CH bond relaxation rates from membrane simulations. Widely used computational approaches offer numerical simplicity but fall short of capturing crucial aspects of the orientation dependence of the dynamics. To circumvent this problem, we introduced a robust framework based on liquid crystal theory which considers explicitly the CH bond motions with respect to the director axis (bilayer normal). Analysis of the orientation dependence of the dynamics shows excellent agreement with experiment, illustrating how the ordering potential affects the calculated relaxation rates. Furthermore, a fit-based resampling of the autocorrelation function of the bond fluctuations validates the new approach for low-temporal resolution data. The recovered relaxation rates indicate that at short timescales, both with and without cholesterol, the local motions of CH bonds describe the bilayer microviscosity and resemble liquid hydrocarbons. Our results establish the critical role of the orientational anisotropy in analysis of membrane simulations, explain fundamental aspects of lipid dynamics, and provide guidelines for extracting information that can be compared to experimental data. STATEMENT OF SIGNIFICANCENuclear magnetic resonance data have been historically used to validate membrane simulations through the average order parameters of the lipid chains. However, the bond dynamics that give rise to this equilibrium bilayer structure have rarely been compared between in vitro and in silico systems despite the availability of substantial experimental data. Here we investigate the logarithmic timescales sampled by the lipid chain motions and confirm a recently developed computational protocol that creates a dynamics-based bridge between simulations and NMR spectroscopy. Our results establish the foundations for validating a relatively unexplored dimension of bilayer behavior and thus have far-reaching applications in membrane biophysics.

biophysics↗

bioRxiv2022

Molecular Simulations and NMR Reveal How Lipid Fluctuations Affect Membrane Mechanics

Lipid bilayers form the main matrix of functional cell membranes, and their dynamics underlie a host of physical and biological processes. Here we show that elastic membrane properties and collective molecular dynamics are related by the mean-square amplitudes (order parameters) and relaxation rates (correlation times) of lipid acyl chain motions. We performed all-atom molecular dynamics simulations of liquid-crystalline bilayers to further interpret available NMR data. Our analysis entailed development of a theoretical framework that allows direct comparison of carbon-hydrogen (CH) bond relaxations as measured by simulations and NMR experiments. The new formalism enables validation of lipid force fields against NMR data by including a fixed bilayer normal (director axis) and restricted anisotropic motion of the CH bonds described by their segmental order parameters. The simulated spectral density of thermally excited CH bond fluctuations exhibited well-defined spin-lattice (Zeeman) relaxations analogous to those measured by solid-state NMR spectroscopy. Their frequency signature could be fit to a simple power-law function, indicative of collective dynamics of a nematic-like nature. The calculated spin-lattice relaxation rates scaled as the squared order parameters of the lipid acyl chains yielding an apparent bending modulus{kappa} C of the bilayer. Our results show a strong correlation with{kappa} C values obtained from solid-state NMR studies of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers with varying amounts of cholesterol as further validated by neutron spin-echo measurements of membrane elasticity. The simulations uncover a critical role of interleaflet coupling in membrane mechanics and thus provide insights into the molecular sites of emerging elastic properties within lipid bilayers. STATEMENT OF SIGNIFICANCEThe lipid make-up of a bilayer determines its measurable properties but how the motions of individual molecules combine to produce these properties remains unclear. By exploiting the synergy between NMR spectroscopy and molecular dynamics (MD) simulations, we show that the lipid dynamics in a bilayer are collective yet segmental in nature and contribute directly to bilayer elasticity. Comparison between MD simulations and NMR entails an improved theoretical framework that allows the two techniques to be directly related. This study thus provides novel insights into the inner workings of lipid membranes while delivering a new tool for validating computational approaches against experimental data.

biophysics↗

bioRxiv2020

Ca2+-dependent mechanism of membrane insertion and destabilization by the SARS-CoV-2 fusion peptide

Cell penetration after recognition of the SARS-CoV-2 virus by the ACE2 receptor, and the fusion of its viral envelope membrane with cellular membranes, are the early steps of infectivity. A region of the Spike protein (S) of the virus, identified as the "fusion peptide" (FP), is liberated at its N-terminal site by a specific cleavage occurring in concert with the interaction of the receptor binding domain of the Spike. Studies have shown that penetration is enhanced by the required binding of Ca2+ ions to the FPs of corona viruses, but the mechanisms of membrane insertion and destabilization remain unclear. We have predicted the preferred positions of Ca2+ binding to the SARS-CoV-2-FP, the role of Ca2+ ions in mediating peptide-membrane interactions, the preferred mode of insertion of the Ca2+-bound SARS-CoV-2-FP and consequent effects on the lipid bilayer from extensive atomistic molecular dynamics (MD) simulations and trajectory analyses. In a systematic sampling of the interactions of the Ca2+-bound peptide models with lipid membranes SARS-CoV-2-FP penetrated the bilayer and disrupted its organization only in two modes involving different structural domains. In one, the hydrophobic residues F833/I834 from the middle region of the peptide are inserted. In the other, more prevalent mode, the penetration involves residues L822/F823 from the LLF motif which is conserved in CoV-2-like viruses, and is achieved by the binding of Ca2+ ions to the D830/D839 and E819/D820 residue pairs. FP penetration is shown to modify the molecular organization in specific areas of the bilayer, and the extent of membrane binding of the SARS-CoV-2 FP is significantly reduced in the absence of Ca2+ ions. These findings provide novel mechanistic insights regarding the role of Ca2+ in mediating SARS-CoV-2 fusion and provide a detailed structural platform to aid the ongoing efforts in rational design of compounds to inhibit SARS-CoV-2 cell entry. STATEMENT OF SIGNIFICANCESARS-CoV-2, the cause of the COVID-19 pandemic, penetrates host cell membranes and uses viral-to-cellular membrane fusion to release its genetic material for replication. Experiments had identified a region termed "fusion peptide" (FP) in the Spike proteins of coronaviruses, as the spearhead in these initial processes, and suggested that Ca2+ is needed to support both functions. Absent structure and dynamics-based mechanistic information these FP functions could not be targeted for therapeutic interventions. We describe the development and determination of the missing information from analysis of extensive MD simulation trajectories, and propose specific Ca2+-dependent mechanisms of SARS-CoV-2-FP membrane insertion and destabilization. These results offer a structure-specific platform to aid the ongoing efforts to use this target for the discovery and/or of inhibitors.

biophysics↗

bioRxiv2020

Direct label-free imaging of nanodomains in biomimetic and biological membranes by cryogenic electron microscopy

The nanoscale organization of biological membranes into structurally and compositionally distinct lateral domains is believed to be central to membrane function. The nature of this organization has remained elusive due to a lack of methods to directly probe nanoscopic membrane features. We show here that cryogenic electron microscopy (cryoEM) can be used to directly image coexisting nanoscopic domains in synthetic and bio-derived membranes without extrinsic probes. Analyzing a series of single-component liposomes composed of synthetic lipids of varying lengths, we demonstrate that cryoEM can distinguish bilayer thickness differences as small as 0.5 [A], comparable to the resolution of small-angle scattering methods. Simulated images from computational models reveal that features in cryoEM images result from a complex interplay between the atomic distribution normal to the plane of the bilayer and imaging parameters. Simulations of phase separated bilayers were used to predict two sources of contrast between coexisting ordered and disordered phases within a single liposome, namely differences in membrane thickness and molecular density. We observe both sources of contrast in biomimetic membranes composed of saturated lipids, unsaturated lipids, and cholesterol. When extended to isolated mammalian plasma membranes, these methods reveal similar nanoscale lateral heterogeneities. The methods reported here for direct, probe-free imaging of nanodomains in unperturbed membranes open new avenues for investigation of nanoscopic membrane organization. SIGNIFICANCEWe have used cryoEM to achieve direct, probe-free imaging of lateral domains in biomimetic lipid membranes under native conditions and to characterize differences in their structures. First, measurements of membrane thickness in laterally uniform single-component membranes show that cryoEM is capable of sub-angstrom resolution of interleaflet membrane thickness. All-atom simulations are used to predict the cryo-EM appearance of submicron domains in vesicles with coexisting liquid domains and these are quantitatively validated by direct imaging of phase separated membranes. We then extend this approach to observe nanoscopic domains in isolated cellular membranes, comprising the first direct imaging of nanodomains in biomembranes.

biophysics↗

bioRxiv2018

Gramicidin increases lipid flip-flop in symmetric and asymmetric lipid vesicles

Unlike most transmembrane proteins, phospholipids can migrate from one leaflet of the membrane to the other. Because this spontaneous lipid translocation (flip-flop) tends to be very slow, cells facilitate the process with enzymes that catalyze the transmembrane movement and thereby regulate the transbilayer lipid distribution. Non-enzymatic membrane-spanning proteins with unrelated primary functions have also been found to accelerate lipid flip-flop in a nonspecific manner and by various hypothesized mechanisms. Using deuterated phospholipids, we examined the acceleration of flip-flop by gramicidin channels which have well-defined structures and known function, features that make them ideal candidates for probing the protein-membrane interactions underlying lipid flip-flop. To study compositionally and isotopically asymmetric proteoliposomes containing gramicidin, we expanded a recently developed protocol for the preparation and characterization of lipid-only asymmetric vesicles. Channel incorporation, conformation, and function were examined with small-angle X-ray scattering, circular dichroism and a stopped-flow spectrofluorometric assay, respectively. As a measure of lipid scrambling we used differential scanning calorimetry to monitor the effect of gramicidin on the melting transition temperatures of the two bilayer leaflets. The two calorimetric peaks of the individual leaflets merged into a single peak over time suggestive of scrambling activity, and the effect of the channel on the transbilayer lipid distribution in both symmetric POPC and asymmetric POPC/DMPC vesicles was quantified from proton NMR measurements. Our results show that gramicidin increases lipid flip-flop in a complex, concentration-dependent manner. To determine the molecular mechanism of the process we used molecular dynamics simulations and further computational analysis of the trajectories to estimate the amount of membrane deformation in the samples. Together, the experimental and computational approaches were found to constitute an effective means for studying the effects of transmembrane proteins on lipid distribution in both symmetric and asymmetric model membranes.

biophysics↗

bioRxiv2018

A new computational method for membrane compressibility: Bilayer mechanical thickness revisited

Because lipid bilayers can bend and stretch in ways similar to thin elastic sheets, physical models of bilayer deformation have utilized mechanical constants such as the moduli for bending rigidity ({kappa}C) and area compressibility (KA). However, the use of these models to quantify the energetics of membrane deformation associated with protein-membrane interactions and the membrane response to stress is often hampered by the shortage of experimental data suitable for the estimation of the mechanical constants of various lipid mixtures. While computational tools such as Molecular Dynamics (MD) simulations can provide alternative means to estimate KA values, current approaches suffer significant technical limitations. Here, we present a novel computational framework that allows for a direct estimation of KA values for individual bilayer leaflets. The theory is based on the concept of elasticity and derives KA from real-space analysis of local thickness fluctuations sampled in MD simulations. We explore and validate the model on a large set of single and multicomponent bilayers of different lipid composition and sizes, simulated at different temperatures. The calculated bilayer compressibility moduli agree with values estimated previously from experiments and those obtained from a standard computational method based on a series of constrained tension simulations. We further validate our framework in a comparison with an existing polymer brush model (PBM) and confirm the PBMs predicted linear relationship with proportionality coefficient of 24 using elastic parameters calculated from the simulation trajectories. The robustness of the results that emerge from the new method allows us to revisit the origins of the bilayer mechanical (compressible) thickness and in particular, its dependence on acyl chain unsaturation and the presence of cholesterol.

biophysics↗