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Understanding oligomerization and aggregation of the amyloid-ß protein is important to elucidate the pathological mechanisms of Alzheimer's disease, and lipid membranes play critical roles in this process. In addition to studies reported by other groups, our group has also reported that the negatively-charged lipid bilayers with a high positive curvature induced α-helix-to-ß-sheet conformational transitions of amyloid-ß-(1-40) upon increase in protein density on the membrane surface and promoted amyloid fibril formation of the protein. Herein, we investigated detailed mechanisms of the conformational transition and oligomer formation of the amyloid-ß protein on the membrane surface. Changes in the fractions of the three protein conformers (free monomer, membrane-bound α-helix-rich conformation, and ß-sheet-rich conformation) were determined from the fluorescent spectral changes of the tryptophan probe in the protein. The helix-to-sheet structural transition on the surface was described by a thermodynamic model of octamer formation driven by entropic forces including hydrophobic interactions. These findings provide useful information for understanding the self-assembly of amyloidogenic proteins on lipid membrane surfaces.
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Péptidos beta-Amiloides , Fosfolípidos , Termodinámica , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Fosfolípidos/química , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Aniones/química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Conformación Proteica en Lámina beta , Estructura Secundaria de Proteína , Humanos , Multimerización de ProteínaRESUMEN
Background: To investigate whether ivermectin inhibits SARS-CoV-2 proliferation in patients with mild-to-moderate COVID-19 using time to a negative COVID-19 reverse transcription-polymerase chain reaction (RT-PCR) test. Methods: CORVETTE-01 was a double-blind, randomized, placebo-controlled study (August 2020-October 2021) conducted in Japan. Overall, 248 patients diagnosed with COVID-19 using RT-PCR were assessed for eligibility. A single oral dose of ivermectin (200 µg/kg) or placebo was administered under fasting. The primary outcome was time to a negative COVID-19 RT-PCR test result for SARS-CoV-2 nucleic acid, assessed using stratified log-rank test and Cox regression models. Results: Overall, 112 and 109 patients were randomized to ivermectin and placebo, respectively; 106 patients from each group were included in the full analysis set (male [%], mean age: 68.9%, 47.9 years [ivermectin]; 62.3%, 47.5 years [placebo]). No significant difference was observed in the occurrence of negative RT-PCR tests between the groups (hazard ratio, 0.96; 95% confidence interval [CI] 0.70-1.32; p = 0.785). Median (95% CI) time to a negative RT-PCR test was 14.0 (13.0-16.0) and 14.0 (12.0-16.0) days for ivermectin and placebo, respectively; 82.1% and 84% of patients achieved negative RT-PCR tests, respectively. Conclusion: In patients with COVID-19, single-dose ivermectin was ineffective in decreasing the time to a negative RT-PCR test. Clinical Trial Registration: ClinicalTrials.gov, NCT04703205.
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The fluid nature of lipid bilayers is indispensable for the dynamic regulation of protein function and membrane morphology in biological membranes. Membrane-spanning domains of proteins interact with surrounding lipids and alter the physical properties of lipid bilayers. However, there is no comprehensive view of the effects of transmembrane proteins on the membrane's physical properties. Here, we investigated the effects of transmembrane peptides with different flip-flop-promoting abilities on the dynamics of a lipid bilayer employing complemental fluorescence and neutron scattering techniques. The quasi-elastic neutron scattering and fluorescence experiments revealed that lateral diffusion of the lipid molecules and the acyl chain motions were inhibited by the inclusion of transmembrane peptides. The neutron spin-echo spectroscopy measurements indicated that the lipid bilayer became more rigid but more compressible and the membrane viscosity increased when the transmembrane peptides were incorporated into the membrane. These results suggest that the inclusion of rigid transmembrane structures hinders individual and collective lipid motions by slowing down lipid diffusion and increasing interleaflet coupling. The present study provides a clue for understanding how the local interactions between lipids and proteins change the collective dynamics of the lipid bilayers, and therefore, the function of biological membranes.
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Membrana Dobles de Lípidos , Fosfatidilcolinas , Membrana Dobles de Lípidos/química , Fosfatidilcolinas/química , Membrana Celular/química , Péptidos/química , Análisis EspectralRESUMEN
The development of cyclic peptides that exhibit pH-sensitive membrane permeation is a promising strategy for tissue-selective drug delivery. We investigated the pH-dependent interactions of designed cyclic peptides bearing noncanonical amino acids of long acidic side chains with lipid membranes, including surface binding, insertion, and translocation across the membrane. As the length of the side chain of acidic amino acid increased, the binding affinity of the peptides to phosphatidylcholine bilayer surfaces decreased, while the pH for the 50% insertion of the peptides into the bilayers increased. The pH for membrane permeation of the peptides increased with the side chain length, resulting in specific membrane permeation at pH â¼6.5. The longer side chain of acidic amino acids improved the maximum rate of membrane permeation at low pH, where both entropic and enthalpic contributions affected the permeation. Our peptide also showed intracellular delivery of cargo molecules into living cells in a pH-dependent manner.
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Membrana Dobles de Lípidos , Péptidos Cíclicos , Péptidos Cíclicos/química , Membrana Dobles de Lípidos/metabolismo , Aminoácidos , Péptidos/química , Concentración de Iones de HidrógenoRESUMEN
Artificial phase-separating (PS) peptides can be used in various applications such as microreactors and drug delivery; however, the design of artificial PS peptides remains a challenge. This can be attributed to the limitation of PS-relevant residues that drive phase separation by interactions of their pairs in short peptides and the difficulty in the design involving interaction with target PS proteins. In this study, we propose a rational method to design artificial PS peptides that satisfy the requirements of liquid droplet formation and co-phase separation with target PS proteins based on the target PS protein sequence. As a proof of concept, we designed five artificial peptides from the model PS protein p53 using this method and confirmed their PS properties using differential interference contrast and fluorescence microscopy. Single-molecule fluorescent tracking demonstrated rapid diffusion of the designed peptides in their droplets compared to that of p53 in p53 droplets. In addition, size-dependent uptake of p53 oligomers was observed in the designed peptide droplets. Large oligomers were excluded from the droplet voids and localized on the droplet surface. The uptake of high-order p53 oligomers into the droplets was enhanced by the elongated linker of the designed peptides. Furthermore, we found that the designed peptide droplets recruited p53 to suppress gel-like aggregate formation. Finally, we discuss aspects that were crucial in the successful design of the artificial PS peptides.
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Péptidos , Proteína p53 Supresora de Tumor , Proteína p53 Supresora de Tumor/metabolismo , Péptidos/química , Secuencia de Aminoácidos , Sistemas de Liberación de MedicamentosRESUMEN
Biological membranes approach one another in various biological phenomena, such as lipid transport at membrane contact sites and membrane fusion. The proximity of two bilayers may cause environmental changes in the interbilayer space and alter the dynamics of lipid molecules. Here, we investigate the structure and dynamics of vesicles aggregated due to the depletion attraction caused by polyethylene glycol (PEG) through static and dynamic small-angle neutron scattering. Manipulation of the interbilayer distance using PEG-conjugated lipids reveals that lipid molecules rapidly transfer between vesicles when the opposing bilayers are within â¼2 nm of each other. This distance corresponds to a region in which water molecules are more structured than in bulk water. Kinetic analysis suggests that the decrease in water entropy is responsible for the progression of lipid transfer. These results provide a basis for understanding the dynamic function of biomembranes in confined regions.
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Membrana Dobles de Lípidos , Agua , Membrana Dobles de Lípidos/química , Cinética , Transporte Biológico , Dispersión del Ángulo Pequeño , Agua/químicaRESUMEN
With development of nanotechnologies, applications exploiting nanospaces such as single-molecule analysis and high-efficiency separation have been reported, and understanding properties of fluid flows in 101 nm to 102 nm scale spaces becomes important. Nanofluidics has provided a platform of nanochannels with defined size and geometry, and revealed various unique liquid properties including higher water viscosity with dominant surface effects in 102 nm spaces. However, experimental investigation of fluid flows in 101 nm spaces is still difficult owing to lack of fabrication procedure for 101 nm nanochannels with smooth walls and precisely controlled geometry. In the present study, we established a top-down fabrication process to realize fused-silica nanochannels with 101 nm scale size, 100 nm roughness and rectangular cross-sectional shape with an aspect ratio of 1. Utilizing a method of mass flowmetry developed by our group, accurate measurements of ultra-low flow rates in sub-100 nm nanochannels with sizes of 70 nm and 100 nm were demonstrated. The results suggested that the viscosity of water in these sub-100 nm nanochannels was approximately 5 times higher than that in the bulk, while that of dimethyl sulfoxide was similar to the bulk value. The obtained liquid permeability in the nanochannels can be explained by a hypothesis of loosely structured liquid phase near the wall generated by interactions between the surface silanol groups and protic solvent molecules. The present results suggest the importance of considering the species of solvent, the surface chemical groups, and the size and geometry of nanospaces when designing nanofluidic devices and membranes.
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Understanding the kinetics of nano-assembly formation is important to elucidate the biological processes involved and develop novel nanomaterials with biological functions. In the present study, we report the kinetic mechanisms of nanofiber formation from a mixture of phospholipids and the amphipathic peptide 18A[A11C], carrying cysteine substitution of the apolipoprotein A-I-derived peptide 18A at residue 11. 18A[A11C] with acetylated N-terminus and amidated C-terminus can associate with phosphatidylcholine to form fibrous aggregates at neutral pH and lipid-to-peptide molar ratio of â¼1, although the reaction pathways of self-assembly remain unclear. Here, the peptide was added to giant 1-palmitoyl-2-oleoyl phosphatidylcholine vesicles to monitor nanofiber formation under fluorescence microscopy. The peptide initially solubilized the lipid vesicles into particles smaller than the resolution of optical microscope, and fibrous aggregates appeared subsequently. Transmission electron microscopy and dynamic light scattering analyses revealed that the vesicle-solubilized particles were spherical or circular, measuring â¼10-20 nm in diameter. The rate of nanofiber formation of 18A with 1,2-dipalmitoyl phosphatidylcholine from the particles was proportional to the square of lipid-peptide concentration in the system, suggesting that the association of particles, accompanied by conformational changes, was the rate-limiting step. Moreover, molecules in the nanofibers could be transferred between aggregates faster than those in the lipid vesicles. These findings provide useful information for the development and control of nano-assembling structures using peptides and phospholipids.
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Nanofibras , Fosfolípidos , Fosfolípidos/metabolismo , Secuencia de Aminoácidos , Péptidos/química , Fosfatidilcolinas , CinéticaRESUMEN
Self-assembling peptides form aggregates with various nanostructures such as spheres, sheets, and fibers and have potential applications in nanomedicine and drug delivery. The alkylation of peptides is a promising strategy for controlling the self-assembly of peptides. In this study, we investigated the thermodynamic properties associated with the aggregation of alkyl-chain-modified self-assembling peptides. The tripeptide sequence, KYF, which has been reported to form fibrous aggregates via self-assembly, was modified with various fatty acids at the N-terminus. The fibrous morphology of the aggregates was observed by transmission electron microscopy and atomic force microscopy. Thioflavin T fluorescence and circular dichroism spectroscopy revealed the formation of ß-sheet structures. The critical micelle concentration and its temperature dependence were determined to obtain the thermodynamic parameters for aggregation. The results showed that the aggregation was an entropy-driven process at low temperatures, whereas it was enthalpy-driven at high temperatures. The negative heat capacity changes for aggregation suggested that hydrophobic interactions were the major driving force for self-assembly. Other entropic and enthalpic interactions were also contributed in part to the self-assembly. We individually identified the contributions of the peptide and alkyl chain moiety to the self-assembly. These contributions can be explained by the theoretical values for the self-assembly of each component. The results of this study provide fundamental insights into the design of self-associating peptides.
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Micelas , Péptidos , Dicroismo Circular , Ácidos Grasos , Interacciones Hidrofóbicas e Hidrofílicas , Péptidos/química , TermodinámicaRESUMEN
Since liquid-liquid phase separation (LLPS) of proteins is governed by their intrinsically disordered regions (IDRs), it can be controlled by LLPS-regulators that bind to the IDRs. The artificial design of LLPS-regulators based on this mechanism can be leveraged in biological and therapeutic applications. However, the fabrication of artificial LLPS-regulators remains challenging. Peptides are promising candidates for artificial LLPS-regulators because of their ability to potentially bind to IDRs complementarily. In this study, we provide a rational peptide design methodology for targeting IDRs based on residue-residue contact energy obtained using molecular dynamics (MD) simulations. This methodology provides rational peptide sequences that function as LLPS regulators. The peptides designed with the MD-based contact energy showed dissociation constants of 35-280 nM for the N-terminal IDR of the tumor suppressor p53, which are significantly lower than the dissociation constants of peptides designed with the conventional 3D structure-based energy, demonstrating the validity of the present peptide design methodology. Importantly, all of the designed peptides enhanced p53 droplet formation. The droplet-forming peptides were converted to droplet-deforming peptides by fusing maltose-binding protein (a soluble tag) to the designed peptides. Thus, the present peptide design methodology for targeting IDRs is useful for regulating droplet formation.
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Proteínas Intrínsecamente Desordenadas , Proteínas Intrínsecamente Desordenadas/química , Simulación de Dinámica Molecular , Péptidos/metabolismo , Fenómenos Físicos , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Understanding how lipid dynamics change with membrane curvature is important given that biological membranes constantly change their curvature and morphology through membrane fusion and endo-/exocytosis. Here, we used time-resolved small-angle neutron scattering and time-resolved fluorescence to characterize the properties and dynamics of phospholipids in vesicles with different curvatures. Dissociation of phospholipids from vesicles required traversing an energy barrier comprising positive enthalpy and negative entropy. However, lipids in membranes with high positive curvature have dense acyl chain packing and loose headgroup packing, leading to hydrophobic hydration due to water penetration into the membrane. These properties were found to lower the hydrophobic hydration enhancement associated with phospholipid dissociation and mitigate the acyl chain packing of lipids adjacent to the space created by the lipid dissociation, resulting in an increase in activation entropy. The results of this study provide important insights into the functions of biomembranes in relation to their dynamic structural changes.
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Membrana Dobles de Lípidos , Fosfolípidos , Membrana Dobles de Lípidos/química , Fusión de Membrana , Neutrones , Fosfolípidos/química , Dispersión del Ángulo PequeñoRESUMEN
The kinetic mechanism of amyloid fibril formation by a peptide fragment containing seven residues of the amyloid-ß protein Aß-(16-22) was investigated. We found that the N- and C-terminal unprotected Aß-(16-22), containing no aggregation nuclei, showed rapid fibrillation within seconds to minutes in a neutral aqueous buffer solution. The fibrillation kinetics were well described by the nucleation-elongation model, suggesting that primary nucleation was the rate-limiting step. On the basis of both experimental and theoretical analyses, the aggregated nucleus was estimated to be composed of 6-7 peptide molecules, wherein the two ß-sheets were associated with their hydrophobic surfaces. Thin fibers with widths of 10-20 nm were formed, which increased their length and thickness, attaining a width of >20 nm over several tens of minutes, probably owing to the lateral association of the fibers. Electrostatic and hydrophobic interactions play important roles in aggregation. These results provide a basis for understanding the fibrillation of short peptides.
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Péptidos beta-Amiloides , Fragmentos de Péptidos , Amiloide/química , Péptidos beta-Amiloides/química , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Fragmentos de Péptidos/química , Agua/químicaRESUMEN
Amphipathic α-helical peptides have been reported to form discoidal particles or nanodiscs with phospholipids, in which a lipid bilayer patch is encircled by peptides. Peptide-based nanodiscs have broad applicability because of their ease of preparation, size flexibility, and structural plasticity. We previously revealed that the nanodiscs formed by apolipoprotein-A-I-derived peptide 18A showed temperature-dependent structural destabilization above the gel-to-liquid-crystalline phase transition temperature of the lipid bilayer. It has been suggested that this destabilization is due to the migration of peptides bound to the edge of the discs to the bilayer surface. In this study, we designed a peptide that could stabilize nanodisc structures against the phase transition of lipid bilayers by disulfide cross-linking of peptides. An 18A-dimer cross-linked by a proline residue, 37pA (Ac-18A-P-18A-CONH2), also showed thermal destabilization of nanodiscs like 18A. However, cross-linking the sides of the two α-helices of the cysteine-substituted analogue 37pA-C2 with disulfide bonds led to the formation of nanodiscs that were more stable to temperature changes. This stabilizing effect was mainly due to the formation of a cyclic 37pA-C2 monomer by intramolecular disulfide cross-linking. These results suggest that the lateral association of two α-helices, which is the basis of the double-belt structure, is an important factor for the implementation of stable nanodiscs. The results of this study will help in development of more stable nanoparticles with membrane proteins in the future.
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Membrana Dobles de Lípidos , Fosfolípidos , Secuencia de Aminoácidos , Disulfuros , Péptidos/química , Fosfolípidos/química , Conformación Proteica en Hélice alfaRESUMEN
It has been demonstrated that peripheral inflammation induces cognitive dysfunction. Several histone deacetylase (HDAC) inhibitors ameliorate cognitive dysfunction in animal models of not only peripheral inflammation but also Alzheimer's disease. However, it is not clear which HDAC expressed in the central nervous system or peripheral tissues is involved in the therapeutic effect of HDAC inhibition on cognitive dysfunction. Hence, the present study investigated the effect of peripheral HDAC inhibition on peripheral inflammation-induced cognitive dysfunction. Suberoylanilide hydroxamic acid (SAHA), a pan-HDAC inhibitor that is mainly distributed in peripheral tissues after intraperitoneal administration, was found to prevent peripheral inflammation-induced cognitive dysfunction. Moreover, pretreatment with SAHA dramatically increased mRNA expression of interleukin-10, an anti-inflammatory cytokine, in peripheral and central tissues and attenuated peripheral inflammation-induced microglial activation in the CA3 region of the hippocampus. Minocycline, a macrophage/microglia inhibitor, also ameliorated cognitive dysfunction. Furthermore, as a result of treatment with liposomal clodronate, depletion of peripheral macrophages partially ameliorated the peripheral inflammation-evoked cognitive dysfunction. Taken together, these findings demonstrate that inhibition of peripheral HDAC plays a critical role in preventing cognitive dysfunction induced by peripheral inflammation via the regulation of anti-inflammatory cytokine production and the inhibition of microglial functions in the hippocampus. Thus, these findings could provide support for inhibition of peripheral HDAC as a novel therapeutic strategy for inflammation-induced cognitive dysfunction.
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Disfunción Cognitiva/tratamiento farmacológico , Inhibidores de Histona Desacetilasas/uso terapéutico , Microglía/efectos de los fármacos , Vorinostat/uso terapéutico , Animales , Disfunción Cognitiva/etiología , Disfunción Cognitiva/metabolismo , Citocinas/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Inflamación/inducido químicamente , Inflamación/complicaciones , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Lipopolisacáridos , Masculino , Ratones , Microglía/metabolismoRESUMEN
Near-field thermophotovoltaic (TPV) power generation has been attracting increasing attention as a promising approach for efficient conversion of heat into electricity with high output power density. Here, we numerically investigate near-field TPV devices with surrounding reflectors for efficient recycling of low-energy photons, which do not contribute to the power generation. We reveal that the conversion efficiency of a near-field TPV system can be drastically increased by introducing a pair of reflectors above and below the system, especially when the two mirrors are not in contact with the emitter and absorber. In addition, we investigate the influence of non-perfect photon recycling on the TPV efficiency and reveal that near-field TPV systems are more robust against the decrease of the reflectivity of the reflectors than the far-field TPV systems.
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Phospholipid transbilayer movement (flip-flop) in the plasma membrane is regulated by membrane proteins to maintain cell homeostasis and interact with other cells. The promotion of flip-flop by phospholipid scramblases causes the loss of membrane lipid asymmetry, which is involved in apoptosis, blood coagulation, and viral infection. Therefore, compounds that can artificially control flip-flop in the plasma membrane are of biological and medical interest. Here, we have developed lipid scrambling transmembrane peptides that can be inserted into the membrane. Time-resolved small-angle neutron scattering measurements revealed that the addition of peptides containing a glutamine residue at the center of the hydrophobic sequence to lipid vesicles induces the flip-flop of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. Peptides without the glutamine residue had no effect on the flip-flop. Because the glutamine-containing peptides exhibited scramblase activity in monomeric form, the polar glutamine residue would be exposed to the hydrocarbon region of the membrane, perturbing the membrane and promoting the lipid flip-flop. These scrambling peptides would be valuable tools to regulate lipid flip-flop in the plasma membrane.
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Liquid droplets of aggregation-prone proteins, which become hydrogels or form amyloid fibrils, are a potential target for drug discovery. In this study, we proposed an experiment-guided protocol for characterizing the design grammar of peptides that can regulate droplet formation and aggregation. The protocol essentially involves investigation of 19 amino acid additives and polymerization of the identified amino acids. As a proof of concept, we applied this protocol to fused in sarcoma (FUS). First, we evaluated 19 amino acid additives for an FUS solution and identified Arg and Tyr as suppressors of droplet formation. Molecular dynamics simulations suggested that the Arg additive interacts with specific residues of FUS, thereby inhibiting the cation-π and electrostatic interactions between the FUS molecules. Second, we observed that Arg polymers promote FUS droplet formation, unlike Arg monomers, by bridging the FUS molecules. Third, we found that the Arg additive suppressed solid aggregate formation of FUS, while Arg polymer enhanced it. Finally, we observed that amyloid-forming peptides induced the conversion of FUS droplets to solid aggregates of FUS. The developed protocol could be used for the primary design of peptides controlling liquid droplets and aggregates of proteins.
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Diseño de Fármacos , Gotas Lipídicas/metabolismo , Péptidos/química , Agregado de Proteínas , Proteína FUS de Unión a ARN/metabolismo , Sitios de Unión , Fenómenos Químicos , Gotas Lipídicas/química , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Estructura Molecular , Péptidos/farmacología , Agregado de Proteínas/efectos de los fármacos , Agregación Patológica de Proteínas , Unión Proteica , Conformación Proteica , Proteína FUS de Unión a ARN/química , Relación Estructura-ActividadRESUMEN
The design of nanoassemblies is an important part of the development of new materials for applications in nanomedicine and biosensors. In our previous study, cysteine substitution of the apolipoprotein A-I-derived peptide 18A at residue 11, 18A[A11C], bound to 1-palmitoyl-2-oleoylphosphatidylcholine to form fibrous aggregates at a lipid-to-peptide molar ratio of ≤2 and a fiber diameter of 10-20 nm. However, the mechanisms underlying the lipid-peptide interactions that enable nanofiber formation remain unclear. Here, we evaluated the phospholipid specificity, concentration dependence, and temperature dependence of the formation of 18A[A11C]-lipid nanofibers. Nanofibers were found to form in the presence of specific phospholipids and have a constant lipid/peptide stoichiometry of 1.2 ± 0.2. Moreover, an increase in the length of the acyl chain in phosphatidylcholines was found to increase the structural stability of the nanofibers. These results indicate that specific molecular interactions between peptides and both the headgroups and acyl chains of phospholipids are involved in nanofiber formation. Furthermore, the formation and disassembly of the nanofibers were reversibly controlled by changes in temperature and concentration. The results of the present study provide an insight into the creation of nanoassembling structures.
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Nanodiscs are discoidal particles in which a lipid bilayer is encircled by amphipathic molecules such as proteins, peptides, or synthetic polymers. The apolipoprotein-A-I-derived peptide 18A is known to form nanodiscs in the presence of phospholipids, but the detailed mechanism of the formation and deformation of these nanodiscs in response to changes in the surrounding environment is not well understood. Here, we investigated the temperature- and composition-dependent structural changes of 18A-phosphatidylcholine complexes using fluorescence spectroscopy, dynamic light scattering, circular dichroism, static 31P NMR, and electron microscopy. We found that the nanodiscs in fast isotropic rotational motion increased in size above the gel-to-liquid-crystalline phase transition temperature of the lipid bilayers, resulting in the formation of enlarged nanodiscs and a lamellar phase. The lamellar phase was found to be oriented along the magnetic field. Further increase in temperature induced the formation of lipid vesicles. These transformations were explained using a transition model based on the migration of the peptide from the rim of the nanodiscs to the liquid-crystalline bilayer phase. The study outcomes provide a basis for understanding the design principles of discoidal nanostructures for structural biology and nanomedicine applications.
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Nanoestructuras , Fosfolípidos , Membrana Dobles de Lípidos , Conformación Molecular , Péptidos , TemperaturaRESUMEN
Sec14, a yeast phosphatidylinositol/phosphatidylcholine transfer protein, functions at the trans-Golgi membranes. It lacks domains involved in protein-protein or protein-lipid interactions and consists solely of the Sec14 domain; hence, the mechanism underlying Sec14 function at proper sites remains unclear. In this study, we focused on the lipid packing of membranes and evaluated its association with in vitro Sec14 lipid transfer activity. Phospholipid transfer assays using pyrene-labelled phosphatidylcholine suggested that increased membrane curvature as well as the incorporation of phosphatidylethanolamine accelerated the lipid transfer. The quantity of membrane-bound Sec14 significantly increased in these membranes, indicating that "packing defects" of the membranes promote the membrane binding and phospholipid transfer of Sec14. Increased levels of phospholipid unsaturation promoted Sec14-mediated PC transfer, but had little effect on the membrane binding of the protein. Our results demonstrate the possibility that the location and function of Sec14 are regulated by the lipid packing states produced by a translocase activity at the trans-Golgi network.