Lipid Rearrangement Research Articles

Hydrophobic Mismatch in the Thylakoid Membrane Regulates Photosynthetic Light Harvesting.

The ability to harvest light effectively in a changing environment is necessary to ensure efficient photosynthesis and crop growth. One mechanism, known as qE, protects photosystem II (PSII) and regulates electron transfer through the harmless dissipation of excess absorbed photons as heat. This process involves reversible clustering of the major light-harvesting complexes of PSII (LHCII) in the thylakoid membrane and relies upon the ΔpH gradient and the allosteric modulator protein PsbS. To date, the exact role of PsbS in the qE mechanism has remained elusive. Here, we show that PsbS induces hydrophobic mismatch in the thylakoid membrane through dynamic rearrangement of lipids around LHCII leading to observed membrane thinning. We found that upon illumination, the thylakoid membrane reversibly shrinks from around 4.3 to 3.2 nm, without PsbS, this response is eliminated. Furthermore, we show that the lipid digalactosyldiacylglycerol (DGDG) is repelled from the LHCII-PsbS complex due to an increase in both the pKa of lumenal residues and in the dipole moment of LHCII, which allows for further conformational change and clustering in the membrane. Our results suggest a mechanistic role for PsbS as a facilitator of a hydrophobic mismatch-mediated phase transition between LHCII-PsbS and its environment. This could act as the driving force to sort LHCII into photoprotective nanodomains in the thylakoid membrane. This work shows an example of the key role of the hydrophobic mismatch process in regulating membrane protein function in plants.

Charge of a transmembrane peptide alters its interaction with lipid membranes

Transmembrane (TM) proteins interact closely with the surrounding membrane lipids. Lipids in the vicinity of TM proteins were reported to have hindered mobility, which has been associated with lipids being caught up in the rough surface of the TM domains. These reports, however, neglect one important factor that largely influences the membrane behavior — electrostatics of the TM peptides that are usually positively charged at their cytosolic end. Here, we study on the example of a neutral and a positively charged WALP peptide, how the charge of a TM peptide influences the membrane. We investigate both its dynamics and mechanics by: (i) time dependent fluorescent shift in combination with classical and FRET generalized polarization to evaluate the mobility of lipids at short and long-range distance from the peptide, (ii) atomic force microscopy to observe the mechanical stability of the peptide-containing membranes, and (iii) molecular dynamics simulations to analyze the peptide-lipid interactions. We show that both TM peptides lower lipid mobility in their closest surroundings. The peptides cause lateral heterogeneity in lipid mobility, which in turn prevents free lipid rearrangement and lowers the membrane ability to seal ruptures after mechanical indentations. Introduction of a positive charge to the peptide largely enhances these effects, affecting the whole membrane. We thus highlight that unspecific peptide-lipid interactions, especially the electrostatics, should not be overlooked as they have a great impact on the mechanics and dynamics of the whole membrane.

Mechanisms for the interaction of the milk fat globule membrane with the plasma membrane of gut epithelial cells

The milk fat globule membrane (MFGM) provides infants and adults with several health benefits. These are not derived solely from its unique composition, but also from arrangement of lipids in the MFGM that, in the case of newborns, could reach the intestine partially intact. Fluorochromes associated with lipid derivatives were used to prove a fusion process between the MFGM and the cellular membrane of differentiated Caco-2 cells. To explore the mechanism of this interaction, incubations of MFGM with Caco-2 cells were carried out in the presence of fusogenic agents or compounds that block other MFGM interaction pathways with cells. Confocal fluorescence microscopy provided visual evidence of the fusion process. Lastly, determination on the lipid profile of cells after their interaction with MFGM indicated a metabolic rearrangement of lipids leading to accumulation of triacylglycerols.

Salivary lipid mediators: Key indexes of inflammation regulation in heart failure disease.

Cardiovascular diseases (CVDs) are the leading cause of premature death and disability in humans and their incidence continues to increase. Oxidative stress and inflammation have been recognized as key pathophysiological factors in cardiovascular events. The targeted modulation of the endogenous mechanisms of inflammation, rather than its simple suppression, will become key in treating chronic inflammatory diseases. A comprehensive characterization of the signalling molecules involved in inflammation, such as endogenous lipid mediators, is thus needed. Here, we propose a powerful MS-based platform for the simultaneous quantitation of sixty salivary lipid mediators in CVD samples. Saliva, which represents a non-invasive and painless alternative to blood, was collected from patients suffering from acute and chronic heart failure (AHF and CHF, respectively), obesity and hypertension. Of all the patients, those with AHF and hypertension showed higher levels of isoprostanoids, which are key indexes of oxidant insult. Compared to the obese population, AHF patients showed lower levels (p<0.02) of antioxidant omega-3 fatty acids, in line with the "malnutrition-inflammation complex syndrome" typical of HF patients. At hospital admission, AHF patients showed significantly higher levels (p<0.001) of omega-3 DPA and lower levels (p<0.04) of lipoxin B4 than CHF patients, suggesting a lipid rearrangement typical of the failing heart during acute decompensation. If confirmed, our results highlight the potential use of lipid mediators as predictive markers of re-acutisation episodes, thus providing opportunities for preventive intervention and a reduction in hospitalizations.

Peptide-membrane binding is not enough to explain bioactivity: A case study

Membrane-active peptides are a promising class of antimicrobial and anticancer therapeutics. For this reason, their molecular mechanisms of action are currently actively investigated. By exploiting Electron Paramagnetic Resonance, we study the membrane interaction of two spin-labeled analogs of the antimicrobial and cytotoxic peptide trichogin GA IV (Tri), with opposite bioactivity: Tri(Api8), able to selectively kill cancer cells, and Tri(Leu4), which is completely nontoxic. In our attempt to determine the molecular basis of their different biological activity, we investigate peptide impact on the lateral organization of lipid membranes, peptide localization and oligomerization, in the zwitter-ionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) model membrane We show that, despite their divergent bioactivity, both peptide analogs (i) are membrane-bound, (ii) display a weak tendency to oligomerization, and (iii) do not induce significant lipid rearrangement. Conversely, literature data show that the parent peptide trichogin, which is cytotoxic without any selectivity, is strongly prone to dimerization and affects the reorganization of POPC membranes. Its dimers are involved in the rotation around the peptide helix, as observed at cryogenic temperatures in the millisecond timescale. Since this latter behavior is not observed for the inactive Tri(Leu4), we propose that for short-length peptides as trichogin oligomerization and molecular motions are crucial for bioactivity, and membrane binding alone is not enough to predict or explain it. We envisage that small changes in the peptide sequence that affect only their ability to oligomerize, or their molecular motions inside the membrane, can tune the peptide activity on membranes of different compositions.

Distinct Cellular Tools of Mild Hyperthermia-Induced Acquired Stress Tolerance in Chinese Hamster Ovary Cells.

Mild stress could help cells to survive more severe environmental or pathophysiological conditions. In the current study, we investigated the cellular mechanisms which contribute to the development of stress tolerance upon a prolonged (0–12 h) fever-like (40 °C) or a moderate (42.5 °C) hyperthermia in mammalian Chinese Hamster Ovary (CHO) cells. Our results indicate that mild heat triggers a distinct, dose-dependent remodeling of the cellular lipidome followed by the expression of heat shock proteins only at higher heat dosages. A significant elevation in the relative concentration of saturated membrane lipid species and specific lysophosphatidylinositol and sphingolipid species suggests prompt membrane microdomain reorganization and an overall membrane rigidification in response to the fluidizing heat in a time-dependent manner. RNAseq experiments reveal that mild heat initiates endoplasmic reticulum stress-related signaling cascades resulting in lipid rearrangement and ultimately in an elevated resistance against membrane fluidization by benzyl alcohol. To protect cells against lethal, protein-denaturing high temperatures, the classical heat shock protein response was required. The different layers of stress response elicited by different heat dosages highlight the capability of cells to utilize multiple tools to gain resistance against or to survive lethal stress conditions.

TMEM16 scramblases thin the membrane to enable lipid scrambling

TMEM16 scramblases dissipate the plasma membrane lipid asymmetry to activate multiple eukaryotic cellular pathways. Scrambling was proposed to occur with lipid headgroups moving between leaflets through a membrane-spanning hydrophilic groove. Direct information on lipid-groove interactions is lacking. We report the 2.3 Å resolution cryogenic electron microscopy structure of the nanodisc-reconstituted Ca2+-bound afTMEM16 scramblase showing how rearrangement of individual lipids at the open pathway results in pronounced membrane thinning. Only the groove’s intracellular vestibule contacts lipids, and mutagenesis suggests scrambling does not require specific protein-lipid interactions with the extracellular vestibule. We find scrambling can occur outside a closed groove in thinner membranes and is inhibited in thicker membranes, despite an open pathway. Our results show afTMEM16 thins the membrane to enable scrambling and that an open hydrophilic pathway is not a structural requirement to allow rapid transbilayer movement of lipids. This mechanism could be extended to other scramblases lacking a hydrophilic groove.

How Does the Spatial Confinement of FtsZ to a Membrane Surface Affect Its Polymerization Properties and Function?

FtsZ is the cytoskeletal protein that organizes the formation of the septal ring and orchestrates bacterial cell division. Its association to the membrane is essential for its function. In this mini-review I will address the question of how this association can interfere with the structure and dynamic properties of the filaments and argue that its dynamics could also remodel the underlying lipid membrane through its activity. Thus, lipid rearrangement might need to be considered when trying to understand FtsZ’s function. This new element could help understand how FtsZ assembly coordinates positioning and recruitment of the proteins forming the septal ring inside the cell with the activity of the machinery involved in peptidoglycan synthesis located in the periplasmic space.

Structure-activity relationship of lipid, cyclic peptide and antigen rearrangement of physically mixed vaccines

Currently there is no approved vaccine to prevent and/or treat group A Streptococcus (GAS) infection. With increasing reports of GAS antibiotic resistance, vaccine adjuvants and targeted delivery systems which induce a strong immune response are a widely acknowledged unmet need. Through extensive structure-activity studies, we investigated a cyclic decapeptide physically mixed with a GAS B cell peptide epitope (J8), a universal T helper epitope (PADRE), and different synthetic lipidic moieties as a conceivable self-adjuvanting GAS vaccine. We explored the structure (orientation)-relationship of the chemically-conjugated B cell epitope and T helper epitope peptide as part of this physically-mixed vaccine. Following in vivo assessment in mice, these cyclic lipopeptide vaccines showed successful induction of J8-specific systemic IgG antibodies when administered subcutaneously without additional adjuvant. Interestingly, an exposed C-terminus of the GAS B cell epitope and a 16-carbon alpha-amino fatty acid lipid was required for strong immunoreactivity, capable of effectively opsonising multiple strains of clinically-isolated GAS bacteria. Physicochemical assessment proved the alpha helix structure of the GAS B cell epitope was retained, impacting particle self-assembly and vaccine immunoreactivity. This study showed the capability for a self-adjuvanting cyclic delivery system to act as a vehicle for the delivery of GAS peptide antigens to treat GAS infection.

Simulation and prediction of membrane fusion dynamics

Membrane fusion is an important process by which biological membranes perform their life activities. Simulations show that the membrane fusion process happens mainly through three pathways, where the Stalk-Pore hypothesis, in which two membranes come into close contact to form a stalk to a hemifusion intermediate, and then the fusion pore opens to achieve completely fusion, is widely accepted, and there exist two free energy barriers that break the current structural steady state for lipid rearrangement. Factors of lipid composition, mechanical environment, protein and ion have regulatory roles in the membrane fusion process by effecting membrane curvature structurally and the free energy barriers from energetic perspective. Meanwhile, many theoretical models, represented by the Helfrich model, have been proposed to predict the membrane fusion process. In this paper, we review the research process of membrane fusion and mainly introduce the dynamics of membrane fusion, regulation factors and typical theoretical models.

Glucosylceramide synthase deficiency in the heart compromises β1-adrenergic receptor trafficking.

Aims Cardiac injury and remodelling are associated with the rearrangement of cardiac lipids. Glycosphingolipids are membrane lipids that are important for cellular structure and function, and cardiac dysfunction is a characteristic of rare monogenic diseases with defects in glycosphingolipid synthesis and turnover. However, it is not known how cardiac glycosphingolipids regulate cellular processes in the heart. The aim of this study is to determine the role of cardiac glycosphingolipids in heart function.Methods and results Using human myocardial biopsies, we showed that the glycosphingolipids glucosylceramide and lactosylceramide are present at very low levels in non-ischaemic human heart with normal function and are elevated during remodelling. Similar results were observed in mouse models of cardiac remodelling. We also generated mice with cardiomyocyte-specific deficiency in Ugcg, the gene encoding glucosylceramide synthase (hUgcg –/– mice). In 9- to 10-week-old hUgcg –/– mice, contractile capacity in response to dobutamine stress was reduced. Older hUgcg –/– mice developed severe heart failure and left ventricular dilatation even under baseline conditions and died prematurely. Using RNA-seq and cell culture models, we showed defective endolysosomal retrograde trafficking and autophagy in Ugcg-deficient cardiomyocytes. We also showed that responsiveness to β-adrenergic stimulation was reduced in cardiomyocytes from hUgcg –/– mice and that Ugcg knockdown suppressed the internalization and trafficking of β1-adrenergic receptors.Conclusions Our findings suggest that cardiac glycosphingolipids are required to maintain β-adrenergic signalling and contractile capacity in cardiomyocytes and to preserve normal heart function.

3D Imaging of Lipid-Guided Vesicle Trafficking Along the Cytoskeleton.

Labeling lipids in vivo is a crucial step in exploring detailed molecular mechanisms coordinating phospholipid signaling and membrane trafficking. Using a lipid-binding domain fused with various fluorescent proteins (FPs) [such as single-colored FP, photoactivated-FP and FPs based on Förster resonance energy transfer (FRET)], lipid biosensors are used to monitor the spatiotemporal distribution, concentration, and dynamic characteristics of targeted lipids. Membrane trafficking is accompanied by rearrangement of membrane lipids and the cytoskeleton. 3D live-cell super-resolution (SR) imaging microscopy detects directional vesicle trafficking events in relation to lipids and the cytoskeleton near the plasma membrane (PM) in 3D space. 3D single-molecule co-tracking (smCo-tracking) provides time-lapse images of 3D colocalization between targeted lipids and vesicles. A dwell time pattern indicates the movement of vesicles towards exocytic sites at the PM. 3D single particle tracking (SPT) could track the overlapping trajectory of vesicles and lipids and display the distance to the cytoskeleton. Lipid biosensors provide a noninvasive and natural labeling for exploring the spatiotemporal dynamics and homeostasis of lipid in vivo. 3D live-cell SR imaging microscopy enables rapid, targeted access, deep imaging within samples at high spatial resolutions for real-time visualization of dynamic biological processes in vivo. 3D smCo-tracking analysis offers simultaneous tracking of different molecules to determine lipid-guided vesicle trafficking in a spatiotemporal organized manner. The 3D SPT technique could provide an accurate estimation of trajectories and spatiotemporal colocalization of vesicles and targeted lipids in 3D space, providing a high-precision visualization and depth track of directional vesicle trafficking. Directional vesicle trafficking is a complex and tightly organized process. Identifying specific lipids and other related regulators will contribute to the exploration of lipid vesicle–cytoskeleton interactions that drive directional delivery of cargos under various stress stimuli, particularly pathogen attacks. For 3D live-cell SR imaging microscopy, how to best balance the imaging speed, spatial-temporal resolution, and volumetric SR imaging capability remains a challenging task. Given that lipid derivatives cannot be genetically encoded, the brighter, more photostable and appropriate fluorophores visualizing the dynamic changes of targeted lipids in concentrations remain to be developed in plant cells. This work is supported by the National Natural Science Foundation of China ( 32030010 , 31900162 , 31770197 , 32000182 ), the Program of Introducing Talents of Discipline to Universities (111 project, B13007 ), and Young Elite Scientists Sponsorship Program by CAST ( 2019QNRC001 ). No interests are declared.

The Synaptotagmin-1 C2B Domain Is a Key Regulator in the Stabilization of the Fusion Pore.

Fusion pores serve as an effective mechanism to connect intracellular organelles and release vesicle contents during exocytosis. A complex lipid rearrangement takes place as membranes approximate, bend, fuse, and establish a traversing water channel to define the fusion pore, linking initially isolated chambers. Thermodynamically, the process is unfavorable and thought to be mediated by specialized proteins. In this work, we have developed a reaction coordinate to induce fusion pores from initially flat and parallel lipid bilayers and we have used it to describe the effects of the synaptotagmin-1 C2B domain during the process. We have obtained free-energy profiles of the whole lipid reorganization in biologically realistic membranes, going from planar and parallel bilayers through stalk hemifusion to water channel formation. Our results point to a lysine-rich polybasic region on synaptotagmin-1 C2B as the key to lipid reorganization control through the formation of phosphatidylinositol bisphosphate clusters that stabilize the fusion pore.

Atg3 promotes Atg8 lipidation via altering lipid diffusion and rearrangement

Atg3-catalyzed transferring of Atg8 to phosphatidylethanolamine (PE) in the phagophore membrane is essential for autophagy. Previous studies have demonstrated that this process requires Atg3 to interact with the phagophore membrane via its N-terminal amphipathic helix. In this study, by using combined biochemical and biophysical approaches, our data showed that in addition to binding to the membranes, Atg3 attenuates lipid diffusion and enriches lipid molecules with smaller headgroup. Our data suggest that Atg3 promotes Atg8 lipidation via altering lipid diffusion and rearrangement.

In vitro and in vivo trans-epidermal water loss evaluation following topical drug delivery systems application for pharmaceutical analysis

The measurement of Trans-Epidermal Water Loss (TEWL) allows to evaluate the integrity of Stratum Corneum Epidermis (SCE) barrier after topical application of colloidal nanocarriers by using a non-invasive method. The temporarily modifications of SCE lipids are important for the passage of colloidal nanocarriers across the skin; this passage causes a modification of TEWL values. Niosomes, ethosomes®, and transfersomes® are used as topical drug delivery systems due to their biopharmaceutical properties, and capability to permeate intact through the SCE. In vitro and in vivo evaluation of TEWL values was studied for niosomes, ethosomes® and transfersomes® in occlusive and non-occlusive conditions. TEWL values in vivo, using healthy human volunteers, are ∼12 g/m2∙× h for all nanoformulations after 72 h, due to the rearrangement of lipids forming the SCE membranes. Conversely, TEWL values of healthy human volunteers, that are topically treated with niosomes, ethosomes® and transfersomes®, in non-occlusive conditions, are ∼20 g/m2∙× h. This data was lower than those obtained in occlusive conditions (∼35 g/m2∙× h). In vitro studies agreed results which are obtained in occlusive conditions using healthy human volunteers. SCE lipids of the skin restore their native structure after 72 h of nanocarrier application. In vitro and in vivo results showed that niosomes, ethosomes®, and transfersomes® interact with the skin in a temporary and reversible mode, and they can be used as suitable colloidal nanocarriers to increase the percutaneous permeation of drugs after topical application without damaging the native structure of the skin.

Enterococcus faecalis Adapts to Antimicrobial Conjugated Oligoelectrolytes by Lipid Rearrangement and Differential Expression of Membrane Stress Response Genes.

Conjugated oligoelectrolytes (COEs) are emerging antimicrobials with broad spectrum activity against Gram positive and Gram negative bacteria as well as fungi. Our previous in vitro evolution studies using Enterococcus faecalis grown in the presence of two related COEs (COE1-3C and COE1-3Py) led to the emergence of mutants (changes in liaF and liaR) with a moderate 4- to16-fold increased resistance to COEs. The contribution of liaF and liaR mutations to COE resistance was confirmed by complementation of the mutants, which restored sensitivity to COEs. To better understand the cellular target of COEs, and the mechanism of resistance to COEs, transcriptional changes associated with resistance in the evolved mutants were investigated in this study. The differentially transcribed genes encoded membrane transporters, in addition to proteins associated with cell envelope synthesis and stress responses. Genes encoding membrane transport proteins from the ATP binding cassette superfamily were the most significantly induced or repressed in COE tolerant mutants compared to the wild type when exposed to COEs. Additionally, differences in the membrane localization of a lipophilic dye in E. faecalis exposed to COEs suggested that resistance was associated with lipid rearrangement in the cell membrane. The membrane adaptation to COEs in EFC3C and EFC3Py resulted in an improved tolerance to bile salt and sodium chloride stress. Overall, this study showed that bacterial cell membranes are the primary target of COEs and that E. faecalis adapts to membrane interacting COE molecules by both lipid rearrangement and changes in membrane transporter activity. The level of resistance to COEs suggests that E. faecalis does not have a specific response pathway to elicit resistance against these molecules and this is supported by the rather broad and diverse suite of genes that are induced upon COE exposure as well as cross-resistance to membrane perturbing stressors.

Correlated protein conformational states and membrane dynamics during attack by pore-forming toxins

Pore-forming toxins (PFTs) are a class of proteins implicated in a wide range of virulent bacterial infections and diseases. These toxins bind to target membranes and subsequently oligomerize to form functional pores that eventually lead to cell lysis. While the protein undergoes large conformational changes on the bilayer, the connection between intermediate oligomeric states and lipid reorganization during pore formation is largely unexplored. Cholesterol-dependent cytolysins (CDCs) are a subclass of PFTs widely implicated in food poisoning and other related infections. Using a prototypical CDC, listeriolysin O (LLO), we provide a microscopic connection between pore formation, lipid dynamics, and leakage kinetics by using a combination of Förster resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) measurements on single giant unilamellar vesicles (GUVs). Upon exposure to LLO, two distinct populations of GUVs with widely different leakage kinetics emerge. We attribute these differences to the existence of oligomeric intermediates, sampling various membrane-bound conformational states of the protein, and their intimate coupling to lipid rearrangement and dynamics. Molecular dynamics simulations capture the influence of various membrane-bound conformational states on the lipid and cholesterol dynamics, providing molecular interpretations to the FRET and FCS experiments. Our study establishes a microscopic connection between membrane binding and conformational changes and their influence on lipid reorganization during PFT-mediated cell lysis. Additionally, our study provides insights into membrane-mediated protein interactions widely implicated in cell signaling, fusion, folding, and other biomolecular processes.

Elucidation of the mechanism and energy barrier for anesthetic triggered membrane fusion in model membranes

Membrane fusion is vital for cellular function and is generally mediated via fusogenic proteins and peptides. The mechanistic details and subsequently the transition state dynamics of membrane fusion will be dependent on the type of the fusogenic agent. We have previously established the potential of general anesthetics as a new class of fusion triggering agents in model membranes. We employed two-photon excitation fluorescence cross-correlation spectroscopy (TPE-FCCS) to report on vesicle association kinetics and steady-state fluorescence dequenching assays to monitor lipid mixing kinetics. Using halothane to trigger fusion in 110 nm diameter dioleoylphosphatidylcholine (DOPC) liposomes, we found that lipid rearrangement towards the formation of the fusion stalk was rate limiting. The activation barrier for halothane induced membrane fusion in 110 nm vesicles was found to be ∼40 kJ mol−1. We calculated the enthalpy and entropy of the transition state to be ∼40 kJ mol−1and ∼180 J mol−1K−1, respectively. We have found that the addition of halothane effectively lowers the energy barrier for membrane fusion in less curved vesicles largely due to entropic advantages.

Formation of tubules and helical ribbons by ceramide phosphoethanolamine-containing membranes

Ceramide phosphoethanolamine (CPE), a major sphingolipid in invertebrates, is crucial for axonal ensheathment in Drosophila. Darkfield microscopy revealed that an equimolar mixture of bovine buttermilk CPE (milk CPE) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (diC18:1 PC) tends to form tubules and helical ribbons, while pure milk CPE mainly exhibits amorphous aggregates and, at low frequency, straight needles. Negative staining electron microscopy indicated that helices and tubules were composed of multilayered 5–10 nm thick slab-like structures. Using different molecular species of PC and CPE, we demonstrated that the acyl chain length of CPE but not of PC is crucial for the formation of tubules and helices in equimolar mixtures. Incubation of the lipid suspensions at the respective phase transition temperature of CPE facilitated the formation of both tubules and helices, suggesting a dynamic lipid rearrangement during formation. Substituting diC18:1 PC with diC18:1 PE or diC18:1 PS failed to form tubules and helices. As hydrated galactosylceramide (GalCer), a major lipid in mammalian myelin, has been reported to spontaneously form tubules and helices, it is believed that the ensheathment of axons in mammals and Drosophila is based on similar physical processes with different lipids.

U-shaped caveolin-1 conformations are tightly regulated by hydrogen bonds with lipids.

The structure and dynamics of a truncated (residues 82-136) caveolin-1 (Cav1) construct having a helix-break-helix motif are explored by both all-atom free energy and molecular dynamics (MD) simulations in an explicit bilayer membrane. Two stable Cav1 conformations with small (LB-Cav1) and large hinge angles (RB-Cav1) between two helices are identified although their relative free energy cannot be reliably estimated due to the sampling issues. RB-Cav1s contain one or two lipids residing between the helices that are hydrogen bonded (h-bonded) to both helices in a multidentate fashion. LB-Cav1s show the helices with mono-dentate lipid h-bond interactions or multidentate interactions limited to a single helix at most. The two conformational states of Cav1 remain their initial state during 2-μs MD simulation, suggesting that there is a significant hidden barrier (other than the insertion depth of Cav1 and its hinge angle) and the Cav1 conformational states are tightly regulated by the h-bonds between Cav1 and lipids along with the associated lipid rearrangement during the course of Cav1 conformational changes. © 2019 Wiley Periodicals, Inc.