Hydrophobic Lipid Core Research Articles

Tailoring Core-Shell Metal Coordination for Smart Seed Coatings in Sustainable Agriculture.

The international agriculture and food security sector is grappling with challenges like low crop yields, soil health deficiencies, and inefficient agrochemical use. The application of smart nanotechnology in agriculture, particularly surface functionalization, holds promise but has limited implementation. Engineered nanomaterials used as seed treatments, known as nanopriming, offer a simple technology to improve crop yield and stress tolerance. In this study, a multicomponent platform called Phelm (Phenolic network with a lipid core and metal coordinated shell) is proposed for encapsulating a commercial plant growth regulator, indole-3 acetic acid (IAA). Phelm comprises a hydrophobic solid lipid core, loaded with IAA, and an outer metal coordinated phenolic shell of tannic acid (TA) and Fe3+. The platform aims to treat seeds with encapsulated IAA, which can be controllably released, as well as protect the germination process at high salt concentrations. Phelm showed a remarkable increase in growth parameters of wheat seeds up to 58.6%, despite being irrigated with high concentrations of saltwater (100 mM). These findings suggest that nanopriming of seeds can effectively increase their efficacy even under abiotic stress conditions, which can drastically improve crop yields. Moreover, we envisage that the Phelm core/shell assembly can encapsulate a wide range of agrochemicals and biostimulants to promote sustainable and smart agricultural practices.

Exploring the Properties of Curved Lipid Membranes: Comparative Analysis of Atomistic and Coarse-Grained Force Fields.

Curvature emerges as a fundamental membrane characteristic crucial for diverse biological processes, including vesicle formation, cell signaling, and membrane trafficking. Increasingly valuable insights into atomistic details governing curvature-dependent membrane properties are provided by computer simulations. Nevertheless, the underlying force field models are conventionally calibrated and tested in relation to experimentally derived parameters of planar bilayers, thereby leaving uncertainties concerning their consistency in reproducing curved lipid systems. In this study we compare the depiction of buckled phosphatidylcholine (POPC) and POPC-cholesterol membranes by four popular force field models. Aside from agreement with respect to general trends in curvature dependence of a number of parameters, we observe a few qualitative differences. Among the most prominent ones is the difference between atomistic and coarse grained force fields in their representation of relative compressibility of the polar headgroup region and hydrophobic lipid core. Through a number of downstream effects, this discrepancy can influence the way in which curvature modulates the behavior of membrane bound proteins depending on the adopted simulation model.

Perilipins: A family of five fat-droplet storing proteins that play a significant role in fat homeostasis.

Lipid droplets are organelles with unique spherical structures. They consist of a hydrophobic neutral lipid core that varies depending on the cell type and tissue. These droplets are surrounded by phospholipid monolayers, along with heterogeneous proteins responsible for neutral lipid synthesis and metabolism. Additionally, there are specialized lipid droplet-associated surface proteins. Recent evidence suggests that proteins from the perilipin family (PLIN) are associated with the surface of lipid droplets and are involved in their formation. These proteins have specific roles in hepatic lipid droplet metabolism, such as protecting the lipid droplets from lipase action and maintaining a balance between lipid storage and utilization in specific cells. Metabolic dysfunction-associated steatotic liver disease (MASLD) is characterized by the accumulation of lipid droplets in more than 5% of the hepatocytes. This accumulation can progress into metabolic dysfunction-associated steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. The accumulation of hepatic lipid droplets in the liver is associated with the progression of MASLD and other diseases such as sarcopenic obesity. Therefore, it is crucial to understand the role of perilipins in this accumulation, as these proteins are key targets for developing novel therapeutic strategies. This comprehensive review aims to summarize the structure and characteristics of PLIN proteins, as well as their pathogenic role in the development of hepatic steatosis and fatty liver diseases.

Solubilization of Phospholipid by Surfactin Leading to Lipid Nanodisc and Fibrous Architecture Formation.

Nanodiscs belong to a category of water-soluble lipid bilayer nanoparticles. In vivo nanodisc platforms are useful for studying isolated membrane proteins in their native lipid environment. Thus, the development of a practical method for nanodisc reconstruction has garnered consider-able research interest. This paper reports the self-assembly of a mixture of bio-derived cyclic peptide, surfactin (SF), and l-α-dimyristoylphosphatidylcholine (DMPC). We found that SF induced the solubilization of DMPC multilamellar vesicles to form their nanodiscs, which was confirmed by size-exclusion chromatography, dynamic light scattering, and transmission electron microscopy analyses. Owing to its amphiphilic nature, the self-assembled structure prevents the exposure of the hydrophobic lipid core to aqueous media, thus embedding ubiquinol (CoQ10) as a hydrophobic model compound within the inner region of the nanodiscs. These results highlight the feasibility of preparing nanodiscs without the need for laborious procedures, thereby showcasing their potential to serve as promising carriers for membrane proteins and various organic compounds. Additionally, the regulated self-assembly of the DMPC/SF mixture led to the formation of fibrous architectures. These results show the potential of this mixture to function as a nanoscale membrane surface for investigating molecular recognition events.

YidC from Escherichia coli Forms an Ion-Conducting Pore upon Activation by Ribosomes.

The universally conserved protein YidC aids in the insertion and folding of transmembrane polypeptides. Supposedly, a charged arginine faces its hydrophobic lipid core, facilitating polypeptide sliding along YidC's surface. How the membrane barrier to other molecules may be maintained is unclear. Here, we show that the purified and reconstituted E. coli YidC forms an ion-conducting transmembrane pore upon ribosome or ribosome-nascent chain complex (RNC) binding. In contrast to monomeric YidC structures, an AlphaFold parallel YidC dimer model harbors a pore. Experimental evidence for a dimeric assembly comes from our BN-PAGE analysis of native vesicles, fluorescence correlation spectroscopy studies, single-molecule fluorescence photobleaching observations, and crosslinking experiments. In the dimeric model, the conserved arginine and other residues interacting with nascent chains point into the putative pore. This result suggests the possibility of a YidC-assisted insertion mode alternative to the insertase mechanism.

Perilipin 3 promotes the formation of membrane domains enriched in diacylglycerol and lipid droplet biogenesis proteins.

Lipid droplets (LDs) serve as intracellular stores of energy-rich neutral lipids. LDs form at discrete sites in the endoplasmic reticulum (ER) and they remain closely associated with the ER during lipogenic growth and lipolytic consumption. Their hydrophobic neutral lipid core is covered by a monolayer of phospholipids, which harbors a specific set of proteins. This LD surface is coated and stabilized by perilipins, a family of soluble proteins that specifically target LDs from the cytosol. We have previously used chimeric fusion proteins between perilipins and integral ER membrane proteins to test whether proteins that are anchored to the ER bilayer could be dragged onto the LD monolayer. Expression of these chimeric proteins induces repositioning of the ER membrane around LDs. Here, we test the properties of membrane-anchored perilipins in cells that lack LDs. Unexpectedly, membrane-anchored perilipins induce expansion and vesiculation of the perinuclear membrane resulting in the formation of crescent-shaped membrane domains that harbor LD-like properties. These domains are stained by LD-specific lipophilic dyes, harbor LD marker proteins, and they transform into nascent LDs upon induction of neutral lipid synthesis. These ER domains are enriched in diacylglycerol (DAG) and in ER proteins that are important for early steps of LD biogenesis, including seipin and Pex30. Formation of the domains in vivo depends on DAG levels, and we show that perilipin 3 (PLIN3) binds to liposomes containing DAG in vitro. Taken together, these observations indicate that perilipin not only serve to stabilize the surface of mature LDs but that they are likely to exert a more active role in early steps of LD biogenesis at ER subdomains enriched in DAG, seipin, and neutral lipid biosynthetic enzymes.

Core-Shell DNA-Cholesterol Nanoparticles Exert Lysosomolytic Activity in African Trypanosomes.

Trypanosoma brucei is the causal infectious agent of African trypanosomiasis in humans and Nagana in livestock. Both diseases are currently treated with a small number of chemotherapeutics, which are hampered by a variety of limitations reaching from efficacy and toxicity complications to drug-resistance problems. Here, we explore the forward design of a new class of synthetic trypanocides based on nanostructured, core-shell DNA-lipid particles. In aqueous solution, the particles self-assemble into micelle-type structures consisting of a solvent-exposed, hydrophilic DNA shell and a hydrophobic lipid core. DNA-lipid nanoparticles have membrane-adhesive qualities and can permeabilize lipid membranes. We report the synthesis of DNA-cholesterol nanoparticles, which specifically subvert the membrane integrity of the T. brucei lysosome, killing the parasite with nanomolar potencies. Furthermore, we provide an example of the programmability of the nanoparticles. By functionalizing the DNA shell with a spliced leader (SL)-RNA-specific DNAzyme, we target a second trypanosome-specific pathway (dual-target approach). The DNAzyme provides a backup to counteract the recovery of compromised parasites, which reduces the risk of developing drug resistance.

PH and lipase-responsive nanocarrier-mediated dual drug delivery system to treat periodontitis in diabetic rats.

Precise and controlled drug delivery to treat periodontitis in patients with diabetes remains a significant clinical challenge. Nanoparticle-based drug delivery systems offer a potential therapeutic strategy; however, the low loading efficiency, non-responsiveness, and single effect of conventional nanoparticles hinder their clinical application. In this study, we designed a novel self-assembled, dual responsive, and dual drug-loading nanocarrier system, which comprised two parts: the hydrophobic lipid core formed by 1, 2-Distearoyl-sn-glycero-3-phosphoethanolamine-Poly (ethylene glycol) (DSPE-PEG) loaded with alpha-lipoic acid (ALA); and a hydrophilic shell comprising a poly (amidoamine) dendrimer (PAMAM) that electrostatically adsorbed minocycline hydrochloride (Mino). This unique design allows the controlled release of antioxidant/ALA under lipase stimulation from periodontal pathogens and antimicrobial/Mino under the low pH of the inflammatory microenvironment. In vivo and in vitro studies confirmed that this dual nanocarrier could inhibit the formation of subgingival microbial colonies while promoting osteogenic differentiation of cells under diabetic pathological conditions, and ameliorated periodontal bone resorption. This effective and versatile drug-delivery strategy has good potential applications to inhibit diabetes-associated periodontal bone loss.

Allosteric activation of Tcell antigen receptor signaling by quaternary structure relaxation.

SummaryThe mechanism of T cell antigen receptor (TCR-CD3) signaling remains elusive. Here, we identify mutations in the transmembrane region of TCRβ or CD3ζ that augment peptide T cell antigen receptor (pMHC)-induced signaling not explicable by enhanced ligand binding, lateral diffusion, clustering, or co-receptor function. Using a biochemical assay and molecular dynamics simulation, we demonstrate that the gain-of-function mutations loosen the interaction between TCRαβ and CD3ζ. Similar to the activating mutations, pMHC binding reduces TCRαβ cohesion with CD3ζ. This event occurs prior to CD3ζ phosphorylation and at 0°C. Moreover, we demonstrate that soluble monovalent pMHC alone induces signaling and reduces TCRαβ cohesion with CD3ζ in membrane-bound or solubilised TCR-CD3. Our data provide compelling evidence that pMHC binding suffices to activate allosteric changes propagating from TCRαβ to the CD3 subunits, reconfiguring interchain transmembrane region interactions. These dynamic modifications could change the arrangement of TCR-CD3 boundary lipids to license CD3ζ phosphorylation and initiate signal propagation.

Therapeutic lithium alters polar head-group region of lipid bilayer and prevents lipid peroxidation in forebrain cortex of sleep-deprived rats

Lithium is regarded as a unique therapeutic agent for the management of bipolar disorder (BD). In efforts to explain the favourable effects of lithium in BD, a wide range of mechanisms was suggested. Among those, the effect of clinically relevant concentrations of lithium on the plasma membrane was extensively studied. However, the biophysical properties of brain membranes isolated from experimental animals exposed to acute, short-term and chronic lithium have not been performed to-date. In this study, we compared the biophysical parameters and level of lipid peroxidation in membranes isolated from forebrain cortex (FBC) of therapeutic lithium-treated and/or sleep-deprived rats. Lithium interaction with FBC membranes was characterized by appropriate fluorescent probes. DPH (1,6-diphenyl-1,3,5-hexatriene) and TMA-DPH (1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulphonate) were used for characterization of the hydrophobic lipid core and Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) for the membrane-water interface. Lipid peroxidation was determined by immunoblot analysis of 4-HNE-(4-hydroxynonenal)-protein adducts. The organization of polar head-group region of FBC membranes, measured by Laurdan generalized polarization, was substantially altered by sleep deprivation and augmented by lithium treatment. Hydrophobic membrane interior characterized by steady-state anisotropy of DPH and TMA-DPH fluorescence was unchanged. Chronic lithium had a protective effect against peroxidative damage of membrane lipids in FBC. In summary, lithium administration at a therapeutic level and/or sleep deprivation as an animal model of mania resulted in changes in rat FBC membrane properties.

LipiSensors: Exploiting Lipid Nanoemulsions to Fabricate Ionophore-Based Nanosensors.

Ionophore-based nanosensors (IBNS) are tools that enable quantification of analytes in complex chemical and biological systems. IBNS methodology is adopted from that of bulk optodes where an ion exchange event is converted to a change in optical output. While valuable, an important aspect for application is the ability to intentionally tune their size with simple approaches, and ensure that they contain compounds safe for application. Lipidots are a platform of size tunable lipid nanoemulsions with a hydrophobic lipid core typically used for imaging and drug delivery. Here, we present LipiSensors as size tunable IBNS by exploiting the Lipidot model as a hydrophobic structural support for the sensing moieties that are traditionally encased in plasticized PVC nanoparticles. The LipiSensors we demonstrate here are sensitive and selective for calcium, reversible, and have a lifetime of approximately one week. By changing the calcium sensing components inside the hydrophobic core of the LipiSensors to those sensitive for oxygen, they are also able to be used as ratiometric O2 sensitive nanosensors via a quenching-based mechanism. LipiSensors provide a versatile, general platform nanosensing with the ability to directly tune the size of the sensors while including biocompatible materials as the structural support by merging sensing approaches with the Lipidot platform.

Hydrophobic Characteristic Is Energetically Preferred for Cysteine in a Model Membrane Protein

The naturally occurring amino acid cysteine has often been implicated with a crucial role in maintaining protein structure and stability. An intriguing duality in the intrinsic hydrophobicity of the cysteine side chain is that it exhibits both polar as well as hydrophobic characteristics. Here, we have utilized a cysteine-scanning mutational strategy on the transmembrane β-barrel PagP to examine the membrane depth-dependent energetic contribution of the free cysteine side chain (thiolate) versus the parent residue at an experimental pH of 9.5 in phosphatidylcholine vesicles. We find that introduction of cysteine causes destabilization at several of the 26 lipid-facing sites of PagP that we mutated in this study. The destabilization is minimal (0.5–1.5 kcal/mol) when the mutation is toward the bilayer midplane, whereas it is higher in magnitude (3.0–5.0 kcal/mol) near the bilayer interface. These observations suggest that cysteine forms more favorable interactions with the hydrophobic lipid core as compared to the amphiphilic water-lipid interface. The destabilizing effect is more pronounced when cysteine replaces the interfacial aromatics, which are known to participate in tertiary interaction networks in transmembrane β-barrels. Our observations from experiments involving the introduction of cysteine at the bilayer midplane further strengthen previous views that the free cysteine side chain does possess strongly apolar characteristics. Additionally, the free energy changes observed upon cysteine incorporation show a depth-dependent correlation with the estimated energetic cost of partitioning derived from reported hydrophobicity scales. Our results and observations from the thermodynamic analysis of the PagP barrel may explain why cysteine, despite possessing a polar sulfhydryl group, tends to behave as a hydrophobic (rather than polar) residue in folded protein structures.

Composite whey protein–cellulose nanocrystals at oil-water interface: Towards delaying lipid digestion

Lipid digestion is an interfacial process that is largely controlled by the adsorption of lipase + colipase + bile salts onto the surface of the emulsified lipid droplets. Therefore, engineering oil-in-water (O/W) interfaces that prevent competitive displacement by bile salts and/or delay the transportation of lipase to the hydrophobic lipid core can be effective strategies to delay lipolysis. In this study, we present such an interface using composite protein-particle system, consisting of whey protein isolate (WPI) (1 wt%) + cellulose nanocrystals (CNCs) (1–3 wt%). Droplet size, microscopy at various length scales (confocal, electron microscopy), ζ-potential and kinetics of fatty acid release were used to assess how the presence of CNCs impacted the microstructural stability of the emulsions in in vitro duodenal conditions (pH 6.8, 37 ○C). Adding CNCs at sufficiently high concentrations (3 wt%) significantly decreased the rate and degree of lipolysis as compared to that of protein-coated emulsion droplets. The principal cause of this altered lipolysis profile was the binding of bile salts by CNCs, which restricted both the interfacial displacement and solubilisation of lipid-digestion products by bile salts. The CNCs can be envisaged to be strongly bound to the protein-coated droplets by virtue of hydrogen bonding with the underlying protein. Furthermore, the ability of the CNCs in the continuous phase to bridge several protein-coated droplets reduced the overall surface area available for the lipolysis. Composite WPI + CNC interface holds promise in designing physiologically relevant emulsions to target satiety or delivery systems for sustained release of lipophilic components.

Conserved Amphipathic Helices Mediate Lipid Droplet Targeting of Perilipins 1–3

Perilipins (PLINs) play a key role in energy storage by orchestrating the activity of lipases on the surface of lipid droplets. Failure of this activity results in severe metabolic disease in humans. Unlike all other lipid droplet-associated proteins, PLINs localize almost exclusively to the phospholipid monolayer surrounding the droplet. To understand how they sense and associate with the unique topology of the droplet surface, we studied the localization of human PLINs in Saccharomyces cerevisiae, demonstrating that the targeting mechanism is highly conserved and that 11-mer repeat regions are sufficient for droplet targeting. Mutations designed to disrupt folding of this region into amphipathic helices (AHs) significantly decreased lipid droplet targeting in vivo and in vitro. Finally, we demonstrated a substantial increase in the helicity of this region in the presence of detergent micelles, which was prevented by an AH-disrupting missense mutation. We conclude that highly conserved 11-mer repeat regions of PLINs target lipid droplets by folding into AHs on the droplet surface, thus enabling PLINs to regulate the interface between the hydrophobic lipid core and its surrounding hydrophilic environment.

Exploring Structural Interactions of Tarantula Toxins with Lipid Membranes using Rosetta and Molecular Dynamics Simulation

Tarantula venom toxins belong to a class of cystine knot peptide toxins that have three disulfide bridges stabilizing the inner core of the toxin structure. These toxins target a wide range of ion channels and modulate physiological activities of channels upon binding. Experimental data indicates that tarantula toxins which target voltage sensors of ion channels interact with cell membrane bilayers and potentially form interactions with channels at the membrane-protein interface. However, how these peptide toxins partition and orient in the membrane are not fully understood. We used Rosetta structural modeling and molecular dynamics simulation, to embed three tarantula toxins, VsTx1, GxTx-1E and SgTx1, into lipid membrane environments. Our results suggest that the three tarantula toxins prefer a similar orientation and depth in the membrane bilayer. We observe that hydrophobic residues on the toxins embed deeper in the hydrophobic lipid core region, basic residues prefer interactions with lipid head groups, and acidic residues are exposed to water environment. This unique orientation of peptide toxins exposes residues critical for interaction with the voltage sensors within the lipid environment, suggesting that toxins may utilize this embedding and orientation to interact favorably with voltage sensors of ion channels.

Lipid droplets and associated proteins in the skin: basic research and clinical perspectives.

Lipid droplets (LDs), the major organelles handling fat storage, comprise a hydrophobic neutral lipid core surrounded by a phospholipid monolayer embedded with a protein miscellany. Although lipids of the stratum corneum are essential for the skin barrier, and progressive lipid accumulation culminating in cell disruption is the hallmark of sebaceous differentiation, only a few studies touched on skin LD and associated proteins so far. Here, after briefly introducing the basic facts about LD and associated proteins, we discuss how forthcoming studies may unveil novel players in skin lipid metabolism and candidate target proteins for treating skin diseases.

Effect of intra-membrane C60 fullerenes on the modulus of elasticity and the mechanical resistance of gel and fluid lipid bilayers.

Penetration and partition of C60 to the lipid bilayer core are both relevant to C60 toxicity, and useful to realise C60 biomedical potential. A key aspect is the effect of C60 on bilayer mechanical properties. Here, we present an experimental study on the mechanical effect of the incorporation of C60 into the hydrophobic core of fluid and gel phase zwitterionic phosphatidylcholine (PC) lipid bilayers. We demonstrate its incorporation inside the hydrophobic lipid core and the effect on the packing of the lipids and the vesicle size using a combination of infrared (IR) spectroscopy, atomic force microscopy (AFM) and laser light scattering. Using AFM we measured the Young's modulus of elasticity (E) of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in the absence (presence) of intra-membranous C60 at 24.5 °C. E of fluid phase supported bilayers is not altered by C60, but E increases with incorporation of C60 in gel phase bilayers. The increase is higher for longer hydrocarbon chains: 1.6 times for DPPC and 2 times for DSPC. However the mechanical resistance of gel phase bilayers of curved bilayered structures decreases with the incorporation of C60. Our combined results indicate that C60 causes a decrease in gel phase lipid mobility, i.e. an increase in membrane viscosity.

Activity and architecture of pyroglutamate-modified amyloid-β (AβpE3-42) pores.

Among the family of Aβ peptides, pyroglutamate-modified Aβ (AβpE) peptides are particularly associated with cytotoxicity in Alzheimer’s disease (AD). They represent the dominant fraction of Aβ oligomers in the brains of AD patients, but their accumulation in the brains of elderly individuals with normal cognition is significantly lower. Accumulation of AβpE plaques precedes the formation of plaques of full-length Aβ (Aβ1-40/42). Most of these properties appear to be associated with the higher hydrophobicity of AβpE as well as an increased resistance to enzymatic degradation. However, the important question of whether AβpE peptides induce pore activity in lipid membranes and their potential toxicity compared with other Aβ pores is still open. Here we examine the activity of AβpE pores in anionic membranes using planar bilayer electrical recording and provide their structures using molecular dynamics simulations. We find that AβpE pores spontaneously induce ionic current across the membrane and have some similar properties to the other previously studied pores of the Aβ family. However, there are also some significant differences. The onset of AβpE3-42 pore activity is generally delayed compared with Aβ1-42 pores. However, once formed, AβpE3-42 pores produce increased ion permeability of the membrane, as indicated by a greater occurrence of higher conductance electrical events. Structurally, the lactam ring of AβpE peptides induces a change in the conformation of the N-terminal strands of the AβpE3-42 pores. While the N-termini of wild-type Aβ1–42 peptides normally reside in the bulk water region, the N-termini of AβpE3-42 peptides tend to reside in the hydrophobic lipid core. These studies provide a first step to an understanding of the enhanced toxicity attributed to AβpE peptides.

Modulation of food texture using controlled heteroaggregation of lipid droplets: Principles and applications

ABSTRACTThis article reviews the development and potential application of novel food‐grade materials based on heteroaggregation of oppositely charged particles. These particles typically consist of a hydrophobic lipid core and an electrically charged emulsifier shell. Heteroaggregation leads to the formation of emulsion‐based products that are highly viscous or gel‐like at much lower droplet concentrations than in nonaggregated systems. Heteroaggregation may therefore be useful for developing food products with novel textural characteristics or for creating reduced‐calorie versions of full fat foods. We give an overview of the principles of heteroaggregation, the relationship between emulsion microstructure and texture, and possible commercial applications of this method. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 130: 3833–3841, 2013

Effect of Phospholipid Composition and Phase on Nanodisc Films at the Solid–Liquid Interface as Studied by Neutron Reflectivity

Nanodiscs are disc-like self-assembled structures formed by phospholipids and amphipatic proteins. The proteins wrap like a belt around the hydrophobic part of the lipids, basically producing nanometer-sized patches of lipid bilayers. The bilayer in the nanodisc constitutes a native-like model of the cell membrane and can act as a nanometer-sized container for functional single membrane proteins. In this study, we present a general nanodisc-based system, intended for structural and functional studies of membrane proteins. In this method, the nanodiscs are aligned at a solid surface, providing the ability to determine the average structure of the film along an axis perpendicular to the interface as measured by neutron reflectivity. The nanodisc film was optimized in terms of nanodisc coverage, reduced film roughness, and stability for time-consuming studies. This was achieved by a systematic variation of the lipid phase, charge, and length of lipid tails. Herein, we show that, although all studied nanodiscs align with their lipid bilayer parallel to the interface, gel-phase DMPC nanodiscs form the most suitable film for future membrane protein studies since they yield a dense irreversibly adsorbed film with low roughness and high stability over time. This may be explained by the appropriate matching between the thickness of the hydrophobic lipid core of gel phase DMPC and the height of the belt protein. Moreover, once formed the gel-phase DMPC nanodiscs film can be heated up to melt the lipid bilayer, thus providing a more biologically friendly environment for membrane proteins.