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1.
Int J Mol Sci ; 22(5)2021 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-33806504

RESUMO

Cholesterol is responsible for the plasticity of plasma membranes and is involved in physiological and pathophysiological responses. Cholesterol homeostasis is regulated by oxysterols, such as 25-hydroxycholesterol. The presence of 25-hydroxycholesterol at the membrane level has been shown to interfere with several viruses' entry into their target cells. We used atomic force microscopy to assess the effect of 25-hydroxycholesterol on different properties of supported lipid bilayers with controlled lipid compositions. In particular, we showed that 25-hydroxycholesterol inhibits the lipid-condensing effects of cholesterol, rendering the bilayers less rigid. This study indicates that the inclusion of 25-hydroxycholesterol in plasma membranes or the conversion of part of their cholesterol content into 25-hydroxycholesterol leads to morphological alterations of the sphingomyelin (SM)-enriched domains and promotes lipid packing inhomogeneities. These changes culminate in membrane stiffness variations.


Assuntos
Membrana Celular/química , Hidroxicolesteróis/química , Colesterol/química , Bicamadas Lipídicas/química , Lipídeos/química , Microscopia de Força Atômica/métodos , Esfingomielinas/química
2.
Int J Mol Sci ; 22(6)2021 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-33799606

RESUMO

The interactions at the atomic level between small molecules and the main components of cellular plasma membranes are crucial for elucidating the mechanisms allowing for the entrance of such small species inside the cell. We have performed molecular dynamics and metadynamics simulations of tryptophan, serotonin, and melatonin at the interface of zwitterionic phospholipid bilayers. In this work, we will review recent computer simulation developments and report microscopic properties, such as the area per lipid and thickness of the membranes, atomic radial distribution functions, angular orientations, and free energy landscapes of small molecule binding to the membrane. Cholesterol affects the behaviour of the small molecules, which are mainly buried in the interfacial regions. We have observed a competition between the binding of small molecules to phospholipids and cholesterol through lipidic hydrogen-bonds. Free energy barriers that are associated to translational and orientational changes of melatonin have been found to be between 10-20 kJ/mol for distances of 1 nm between melatonin and the center of the membrane. Corresponding barriers for tryptophan and serotonin that are obtained from reversible work methods are of the order of 10 kJ/mol and reveal strong hydrogen bonding between such species and specific phospholipid sites. The diffusion of tryptophan and melatonin is of the order of 10-7 cm2/s for the cholesterol-free and cholesterol-rich setups.


Assuntos
1,2-Dipalmitoilfosfatidilcolina/química , Colesterol/química , Dimiristoilfosfatidilcolina/química , Melatonina/química , Serotonina/química , Triptofano/química , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Colesterol/metabolismo , Dimiristoilfosfatidilcolina/metabolismo , Ligação de Hidrogênio , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Melatonina/metabolismo , Simulação de Dinâmica Molecular , Serotonina/metabolismo , Soluções , Eletricidade Estática , Termodinâmica , Triptofano/metabolismo , Água/química
3.
Int J Mol Sci ; 22(5)2021 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-33806280

RESUMO

Amphiphilic diisobutylene/maleic acid (DIBMA) copolymers extract lipid-encased membrane proteins from lipid bilayers in a detergent-free manner, yielding nanosized, discoidal DIBMA lipid particles (DIBMALPs). Depending on the DIBMA/lipid ratio, the size of DIBMALPs can be broadly varied which makes them suitable for the incorporation of proteins of different sizes. Here, we examine the influence of the DIBMALP sizes and the presence of protein on the dynamics of encased lipids. As shown by a set of biophysical methods, the stability of DIBMALPs remains unaffected at different DIBMA/lipid ratios. Coarse-grained molecular dynamics simulations confirm the formation of viable DIBMALPs with an overall size of up to 35 nm. Electron paramagnetic resonance spectroscopy of nitroxides located at the 5th, 12th or 16th carbon atom positions in phosphatidylcholine-based spin labels reveals that the dynamics of enclosed lipids are not altered by the DIBMALP size. The presence of the membrane protein sensory rhodopsin II from Natronomonas pharaonis (NpSRII) results in a slight increase in the lipid dynamics compared to empty DIBMALPs. The light-induced photocycle shows full functionality of DIBMALPs-embedded NpSRII and a significant effect of the protein-to-lipid ratio during preparation on the NpSRII dynamics. This study indicates a possible expansion of the applicability of the DIBMALP technology on studies of membrane protein-protein interaction and oligomerization in a constraining environment.


Assuntos
Halorrodopsinas/química , Bicamadas Lipídicas/química , Rodopsinas Sensoriais/química , Alcenos/química , Fenômenos Biofísicos , Dimiristoilfosfatidilcolina/química , Espectroscopia de Ressonância de Spin Eletrônica , Halobacteriaceae/química , Halobacteriaceae/efeitos da radiação , Halorrodopsinas/efeitos da radiação , Maleatos/química , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Simulação de Dinâmica Molecular , Nanopartículas/química , Nanopartículas/ultraestrutura , Tamanho da Partícula , Processos Fotoquímicos , Rodopsinas Sensoriais/efeitos da radiação , Marcadores de Spin
4.
Molecules ; 26(6)2021 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-33809519

RESUMO

Membrane-scaffolding proteins (MSPs) derived from apolipoprotein A-1 have become a versatile tool in generating nano-sized discoidal membrane mimetics (nanodiscs) for membrane protein research. Recent efforts have aimed at exploiting their controlled lipid protein ratio and size distribution to arrange membrane proteins in regular supramolecular structures for diffraction studies. Thereby, direct membrane protein crystallization, which has remained the limiting factor in structure determination of membrane proteins, would be circumvented. We describe here the formation of multimers of membrane-scaffolding protein MSP1D1-bounded nanodiscs using the thiol reactivity of engineered cysteines. The mutated positions N42 and K163 in MSP1D1 were chosen to support chemical modification as evidenced by fluorescent labeling with pyrene. Minimal interference with the nanodisc formation and structure was demonstrated by circular dichroism spectroscopy, differential light scattering and size exclusion chromatography. The direct disulphide bond formation of nanodiscs formed by the MSP1D1_N42C variant led to dimers and trimers with low yield. In contrast, transmission electron microscopy revealed that the attachment of oligonucleotides to the engineered cysteines of MSP1D1 allowed the growth of submicron-sized tracts of stacked nanodiscs through the hybridization of nanodisc populations carrying complementary strands and a flexible spacer.


Assuntos
DNA/química , Bicamadas Lipídicas/química , Proteínas de Membrana/química , Nanoestruturas/química , Sequência de Aminoácidos , Apolipoproteína A-I/química , Microscopia Eletrônica de Transmissão/métodos , Fosfolipídeos/química
5.
Int J Mol Sci ; 22(6)2021 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-33799744

RESUMO

Lasioglossin III (LL-III) is a cationic antimicrobial peptide derived from the venom of the eusocial bee Lasioglossum laticeps. LL-III is extremely toxic to both Gram-positive and Gram-negative bacteria, and it exhibits antifungal as well as antitumor activity. Moreover, it shows low hemolytic activity, and it has almost no toxic effects on eukaryotic cells. However, the molecular basis of the LL-III mechanism of action is still unclear. In this study, we characterized by means of calorimetric (DSC) and spectroscopic (CD, fluorescence) techniques its interaction with liposomes composed of a mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-rac-phosphoglycerol (POPG) lipids as a model of the negatively charged membrane of pathogens. For comparison, the interaction of LL-III with the uncharged POPC liposomes was also studied. Our data showed that LL-III preferentially interacted with anionic lipids in the POPC/POPG liposomes and induces the formation of lipid domains. Furthermore, the leakage experiments showed that the peptide could permeabilize the membrane. Interestingly, our DSC results showed that the peptide-membrane interaction occurs in a non-disruptive manner, indicating an intracellular targeting mode of action for this peptide. Consistent with this hypothesis, our gel-retardation assay experiments showed that LL-III could interact with plasmid DNA, suggesting a possible intracellular target.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Abelhas/metabolismo , Membrana Celular/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Animais , Peptídeos Catiônicos Antimicrobianos/análise , Calorimetria/métodos , Membrana Celular/química , Permeabilidade da Membrana Celular/efeitos dos fármacos , Bicamadas Lipídicas/química , Lipossomos/química , Fosfatidilcolinas/química , Fosfatidilgliceróis/química , Proteínas Citotóxicas Formadoras de Poros/análise , Espectrofotometria/métodos
6.
Nat Commun ; 12(1): 2202, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33850135

RESUMO

Artificial native-like lipid bilayer systems constructed from phospholipids assembling into unilamellar liposomes allow the reconstitution of detergent-solubilized transmembrane proteins into supramolecular lipid-protein assemblies called proteoliposomes, which mimic cellular membranes. Stabilization of these complexes remains challenging because of their chemical composition, the hydrophobicity and structural instability of membrane proteins, and the lability of interactions between protein, detergent, and lipids within micelles and lipid bilayers. In this work we demonstrate that metastable lipid, protein-detergent, and protein-lipid supramolecular complexes can be successfully generated and immobilized within zeolitic-imidazole framework (ZIF) to enhance their stability against chemical and physical stressors. Upon immobilization in ZIF bio-composites, blank liposomes, and model transmembrane metal transporters in detergent micelles or embedded in proteoliposomes resist elevated temperatures, exposure to chemical denaturants, aging, and mechanical stresses. Extensive morphological and functional characterization of the assemblies upon exfoliation reveal that all these complexes encapsulated within the framework maintain their native morphology, structure, and activity, which is otherwise lost rapidly without immobilization.


Assuntos
Detergentes/química , Exoesqueleto Energizado , Imobilização/métodos , Bicamadas Lipídicas/química , Proteínas de Membrana/química , Membrana Celular , ATPases Transportadoras de Cobre , Proteínas de Escherichia coli , Cinética , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana/metabolismo , Micelas , Fosfolipídeos , Proteolipídeos , Espalhamento de Radiação , Lipossomas Unilamelares , Difração de Raios X
7.
ACS Appl Mater Interfaces ; 13(5): 6739-6747, 2021 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-33522221

RESUMO

Multisomes are multicompartmental structures formed by a lipid-stabilized network of aqueous droplets, which are contained by an outer oil phase. These biomimetic structures are emerging as a versatile platform for soft matter and synthetic biology applications. While several methods for producing multisomes have been described, including microfluidic techniques, approaches for generating biocompatible, monodisperse multisomes in a reproducible manner remain challenging to implement due to low throughput and complex device fabrication. Here, we report on a robust method for the dynamically controlled generation of multisomes with controllable sizes and high monodispersity from lipid-based double emulsions. The described microfluidic approach entails the use of three different phases forming a water/oil/water (W/O/W) double emulsion stabilized by lipid layers. We employ a gradient of glycerol concentration between the inner core and outer phase to drive the directed osmosis, allowing the swelling of lamellar lipid layers resulting in the formation of small aqueous daughter droplets at the interface of the inner aqueous core. By adding increasing concentrations of glycerol to the outer aqueous phase and subsequently varying the osmotic gradient, we show that key structural parameters, including the size of the internal droplets, can be specifically controlled. Finally, we show that this approach can be used to generate multisomes encapsulating small-molecule cargo, with potential applications in synthetic biology, drug delivery, and as carriers for active materials in the food and cosmetics industries.


Assuntos
Lipídeos/química , Emulsões/síntese química , Emulsões/química , Bicamadas Lipídicas/química , Óleos/química , Tamanho da Partícula , Propriedades de Superfície , Água/química
8.
Molecules ; 26(3)2021 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-33573083

RESUMO

Collagen contains hydroxyproline (Hyp), which is a unique amino acid. Three collagen-derived small peptides (Gly-Pro-Hyp, Pro-Hyp, and Gly-Hyp) interacting across a lipid bilayer (POPC model membrane) for cellular uptakes of these collagen-derived small peptides were studied using accelerated molecular dynamics simulation. The ligands were investigated for their binding modes, hydrogen bonds in each coordinate frame, and mean square displacement (MSD) in the Z direction. The lipid bilayers were evaluated for mass and electron density profiles of the lipid molecules, surface area of the head groups, and root mean square deviation (RMSD). The simulation results show that hydrogen bonding between the small collagen peptides and plasma membrane plays a significant role in their internalization. The translocation of the small collagen peptides across the cell membranes was shown. Pro-Hyp laterally condensed the membrane, resulting in an increase in the bilayer thickness and rigidity. Perception regarding molecular behaviors of collagen-derived peptides within the cell membrane, including their interactions, provides the novel design of specific bioactive collagen peptides for their applications.


Assuntos
Colágeno/química , Bicamadas Lipídicas/química , Peptídeos/química , Sequência de Aminoácidos/genética , Transporte Biológico/genética , Dicroísmo Circular , Colágeno/genética , Simulação por Computador , Dipeptídeos/química , Dipeptídeos/genética , Ligação de Hidrogênio/efeitos dos fármacos , Hidroxiprolina/química , Peptídeos/genética , Ligação Proteica/genética , Conformação Proteica
9.
Nat Struct Mol Biol ; 28(3): 258-267, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33633398

RESUMO

G-protein-coupled receptors (GPCRs) are the largest superfamily of transmembrane proteins and the targets of over 30% of currently marketed pharmaceuticals. Although several structures have been solved for GPCR-G protein complexes, few are in a lipid membrane environment. Here, we report cryo-EM structures of complexes of neurotensin, neurotensin receptor 1 and Gαi1ß1γ1 in two conformational states, resolved to resolutions of 4.1 and 4.2 Å. The structures, determined in a lipid bilayer without any stabilizing antibodies or nanobodies, reveal an extended network of protein-protein interactions at the GPCR-G protein interface as compared to structures obtained in detergent micelles. The findings show that the lipid membrane modulates the structure and dynamics of complex formation and provide a molecular explanation for the stronger interaction between GPCRs and G proteins in lipid bilayers. We propose an allosteric mechanism for GDP release, providing new insights into the activation of G proteins for downstream signaling.


Assuntos
Microscopia Crioeletrônica , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/ultraestrutura , Bicamadas Lipídicas , Nanoestruturas/química , Receptores de Neurotensina/metabolismo , Receptores de Neurotensina/ultraestrutura , Regulação Alostérica , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/ultraestrutura , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/ultraestrutura , Subunidades gama da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/ultraestrutura , Guanosina Difosfato/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/química , Humanos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Micelas , Modelos Moleculares , Neurotensina/química , Neurotensina/metabolismo , Conformação Proteica , Receptores de Neurotensina/química , Transdução de Sinais
10.
Nature ; 590(7846): 509-514, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33568813

RESUMO

Mechanosensitive channels sense mechanical forces in cell membranes and underlie many biological sensing processes1-3. However, how exactly they sense mechanical force remains under investigation4. The bacterial mechanosensitive channel of small conductance, MscS, is one of the most extensively studied mechanosensitive channels4-8, but how it is regulated by membrane tension remains unclear, even though the structures are known for its open and closed states9-11. Here we used cryo-electron microscopy to determine the structure of MscS in different membrane environments, including one that mimics a membrane under tension. We present the structures of MscS in the subconducting and desensitized states, and demonstrate that the conformation of MscS in a lipid bilayer in the open state is dynamic. Several associated lipids have distinct roles in MscS mechanosensation. Pore lipids are necessary to prevent ion conduction in the closed state. Gatekeeper lipids stabilize the closed conformation and dissociate with membrane tension, allowing the channel to open. Pocket lipids in a solvent-exposed pocket between subunits are pulled out under sustained tension, allowing the channel to transition to the subconducting state and then to the desensitized state. Our results provide a mechanistic underpinning and expand on the 'force-from-lipids' model for MscS mechanosensation4,11.


Assuntos
Microscopia Crioeletrônica , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/ultraestrutura , Escherichia coli/química , Canais Iônicos/metabolismo , Canais Iônicos/ultraestrutura , Membranas Artificiais , Fosfatidilcolinas/metabolismo , Detergentes/farmacologia , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Interações Hidrofóbicas e Hidrofílicas , Canais Iônicos/química , Canais Iônicos/genética , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Mecanotransdução Celular/efeitos dos fármacos , Modelos Moleculares , Mutação , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Fosfatidilcolinas/química , Fosfatidilcolinas/farmacologia , Conformação Proteica/efeitos dos fármacos , beta-Ciclodextrinas/farmacologia
11.
Phys Chem Chem Phys ; 23(3): 2117-2125, 2021 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-33437978

RESUMO

Complexin-1 (Cpx) and α-synuclein (α-Syn) are involved in neurotransmitter release through an interaction with synaptic vesicles (SVs). Recent studies demonstrated that Cpx and α-Syn preferentially associate with highly curved membranes, like SVs, to correctly position them for fusion. Here, based on recent experimental results, to further propose a possible explanation for this mechanism, we performed in silico simulations probing interactions between Cpx or α-Syn and membranes of varying curvature. We found that the preferential association is attributed to smaller, curved membranes containing more packing defects that expose hydrophobic acyl tails, which may favorably interact with hydrophobic residues of Cpx and α-Syn. The number of membrane defects is proportional to the curvature and the size can be regulated by cholesterol.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Bicamadas Lipídicas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Vesículas Sinápticas/metabolismo , alfa-Sinucleína/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/química , Colesterol/química , Ligação de Hidrogênio , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Proteínas do Tecido Nervoso/química , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Fosfatidilserinas/química , Ligação Proteica , Vesículas Sinápticas/química , alfa-Sinucleína/química
12.
Arch Biochem Biophys ; 699: 108751, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33421380

RESUMO

Antimicrobial and cell-penetrating peptides have been the object of extensive studies for more than 60 years. Initially these two families were studied separately, and more recently parallels have been drawn. These studies have given rise to numerous methodological developments both in terms of observation techniques and membrane models. This review presents some of the most recent original and innovative developments in this field, namely droplet interface bilayers (DIBs), new fluorescence approaches, force measurements, and photolabelling.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Membrana Celular/metabolismo , Peptídeos Penetradores de Células/metabolismo , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/química , Membrana Celular/química , Peptídeos Penetradores de Células/química , Corantes Fluorescentes/química , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Microscopia de Fluorescência/métodos , Marcadores de Fotoafinidade/química , Espectrometria de Fluorescência/métodos
13.
ACS Appl Mater Interfaces ; 13(2): 2145-2164, 2021 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-33417432

RESUMO

Oligonucleotide-based probes offer the highest spatial resolution, force sensitivity, and molecular specificity for cellular tension sensing and have been developed to measure a variety of molecular forces mediated by individual receptors in T cells, platelets, fibroblasts, B-cells, and immortalized cancer cell lines. These fluorophore-oligonucleotide conjugate probes are designed with a stem-loop structure that engages cell receptors and reversibly unfolds due to mechanical strain. With the growth of recent work bridging molecular mechanobiology and biomaterials, there is a need for a detailed spectroscopic analysis of DNA tension probes that are used for cellular imaging. In this manuscript, we conducted an analysis of 19 DNA hairpin-based tension probe variants using molecular dynamics simulations, absorption spectroscopy, and fluorescence imaging (epifluorescence and fluorescence lifetime imaging microscopy). We find that tension probes are highly sensitive to their molecular design, including donor and acceptor proximity and pairing, DNA stem-loop structure, and conjugation chemistry. We demonstrate the impact of these design features using a supported lipid bilayer model of podosome-like adhesions. Finally, we discuss the requirements for tension imaging in various biophysical contexts and offer a series of experimental recommendations, thus providing a guide for the design and application of DNA hairpin-based molecular tension probes.


Assuntos
Corantes Fluorescentes/química , Bicamadas Lipídicas/química , Sondas de Oligonucleotídeos/química , Animais , Fenômenos Biomecânicos , Adesão Celular , Transferência Ressonante de Energia de Fluorescência/métodos , Integrinas/análise , Mecanotransdução Celular , Camundongos , Microscopia de Fluorescência/métodos , Modelos Moleculares , Simulação de Dinâmica Molecular , Células NIH 3T3 , Imagem Óptica/métodos , Resistência à Tração
14.
Nat Commun ; 12(1): 172, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33420032

RESUMO

The dimeric transporter, EmrE, effluxes polyaromatic cationic drugs in a proton-coupled manner to confer multidrug resistance in bacteria. Although the protein is known to adopt an antiparallel asymmetric topology, its high-resolution drug-bound structure is so far unknown, limiting our understanding of the molecular basis of promiscuous transport. Here we report an experimental structure of drug-bound EmrE in phospholipid bilayers, determined using 19F and 1H solid-state NMR and a fluorinated substrate, tetra(4-fluorophenyl) phosphonium (F4-TPP+). The drug-binding site, constrained by 214 protein-substrate distances, is dominated by aromatic residues such as W63 and Y60, but is sufficiently spacious for the tetrahedral drug to reorient at physiological temperature. F4-TPP+ lies closer to the proton-binding residue E14 in subunit A than in subunit B, explaining the asymmetric protonation of the protein. The structure gives insight into the molecular mechanism of multidrug recognition by EmrE and establishes the basis for future design of substrate inhibitors to combat antibiotic resistance.


Assuntos
Antiporters/química , Antiporters/efeitos dos fármacos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/efeitos dos fármacos , Bicamadas Lipídicas/química , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Antibacterianos/química , Antibacterianos/farmacologia , Sítios de Ligação , Transporte Biológico/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Escherichia coli/metabolismo , Simulação de Dinâmica Molecular , Conformação Proteica
15.
J Phys Chem Lett ; 12(5): 1384-1389, 2021 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-33508197

RESUMO

One of the key parameters required to identify effective drugs is membrane permeability, as a compound intended for an intracellular target with poor permeability will have low efficacy. In this paper, we leverage a computational approach recently developed by our group to study the interactions between nanoparticles and mammalian membranes to study the time of entry of a variety of drugs into the viral envelope of coronavirus as well as cellular organelles. Using a combination of all-atoms molecular dynamics simulations and statistical analysis, we consider both drug characteristics and membrane properties to determine the behavior of 79 drugs and their interactions with the viral envelope, composed of the membrane and spike protein, as well as five other membranes that correspond to various mammalian compartments (lysosome, plasma, Golgi, mitochondrial, and endoplasmic reticulum membranes). The results highlight important trends that can be exploited for drug design, from the relatively high permeability of the viral envelope and the effect of transmembrane proteins, to the differences in permeability between organelles. When compared with bioavailability data present in the literature, the model results suggest a negative correlation between time of permeation and bioavailability of promising drugs. The method is general and flexible and can be employed for a variety of molecules, from small drugs to small nanoparticles, as well to a variety of biological membranes. Overall, the results indicate that this model can contribute to the identification of successful drugs as it predicts the ability of compounds to reach both intended and unintended intracellular targets.


Assuntos
Antivirais/metabolismo , /efeitos dos fármacos , Sítios de Ligação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Bicamadas Lipídicas/química , Glicoproteínas de Membrana/química , Modelos Biológicos , Simulação de Dinâmica Molecular , Nanopartículas/química , Tamanho da Partícula , Permeabilidade , Ligação Proteica , Solubilidade , Glicoproteína da Espícula de Coronavírus/metabolismo , /efeitos dos fármacos
16.
Methods Mol Biol ; 2251: 143-156, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33481237

RESUMO

It is now clear that organelles of a mammalian cell can be distinguished by phospholipid profiles, both as ratios of common phospholipids and by the absence or presence of certain phospholipids. Organelle-specific phospholipids can be used to provide a specific shape and fluidity to the membrane and/or used to recruit and/or traffic proteins to the appropriate subcellular location and to restrict protein function to this location. Studying the interactions of proteins with specific phospholipids using soluble derivatives in isolation does not always provide useful information because the context in which the headgroups are presented almost always matters. Our laboratory has shown this circumstance to be the case for a viral protein binding to phosphoinositides in solution and in membranes. The system we have developed to study protein-phospholipid interactions in the context of a membrane benefits from the creation of tailored membranes in a channel of a microfluidic device, with a fluorescent lipid in the membrane serving as an indirect reporter of protein binding. This system is amenable to the study of myriad interactions occurring at a membrane surface as long as a net change in surface charge occurs in response to the binding event of interest.


Assuntos
Membranas/metabolismo , Técnicas Analíticas Microfluídicas/métodos , Fosfolipídeos/análise , Animais , Humanos , Dispositivos Lab-On-A-Chip , Bicamadas Lipídicas/química , Microfluídica/métodos , Fosfatidilinositóis/metabolismo , Fosfolipídeos/química , Ligação Proteica/fisiologia , Proteínas/metabolismo
17.
Methods Mol Biol ; 2251: 215-223, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33481243

RESUMO

Phosphoinositides play important roles in the regulation of protein recruitment at specialized membrane domains, protein activity, and membrane dynamics. Phosphoinositide-protein interplay occurs via multiple mechanisms and proteins associate with membranes through different binding patterns. Determinations of membrane-binding mode and membrane penetration depth of proteins in lipid bilayer are thus important steps in characterizing the molecular mechanisms of membrane-protein interactions. Here, we show two standard in vitro assays using liposomes, diphenylhexatriene (DPH) anisotropy, and fluorescence quenching by brominated lipids to determine membrane penetration of proteins into lipid bilayer. These methods will provide useful tools to study membrane-protein association and uncover molecular details of protein-lipid interplay, which are important for understanding biological functions of membrane-associated proteins and membrane dynamics.


Assuntos
Polarização de Fluorescência/métodos , Fluidez de Membrana/fisiologia , Espectrometria de Fluorescência/métodos , Animais , Difenilexatrieno/química , Fluorescência , Corantes Fluorescentes/química , Humanos , Bicamadas Lipídicas/química , Lipossomos/química , Proteínas de Membrana/análise , Proteínas de Membrana/química , Membranas/química , Fosfatidilcolinas/química , Fosfatidilinositóis/análise , Fosfatidilinositóis/química
18.
Methods Mol Biol ; 2207: 299-312, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33113143

RESUMO

Differential scanning calorimetry (DSC) is a well-established technique, suitable to monitor the interactions that may take place among the drug delivery systems of liposomes and the potential bioactive molecules that are incorporated inside them. Moreover, the DSC technique is considered to be a useful tool to characterize the thermal behavior of lipidic bilayers in the absence and presence of drugs and to highlight parameters, such as the cooperativity between the lipids and the guest molecules (i.e. drugs, polymers, dendrimers), providing also a prediction of the behavior of potential future drug delivery liposomal platforms. In this study, a protocol for DSC measurements on liposomal systems with incorporated guest molecules is described.


Assuntos
1,2-Dipalmitoilfosfatidilcolina/química , Dendrímeros/química , Bicamadas Lipídicas/química , Avaliação de Medicamentos , Lipossomos
19.
Methods Mol Biol ; 2186: 19-32, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32918727

RESUMO

Membrane protein pores have demonstrated applications in nanopore technology. Previous studies have mostly focused on ß-barrel protein pores, whereas α-helix-based transmembrane protein pores are rarely explored in nanopore applications. Here, we developed a synthetic transmembrane peptide pore built entirely from short synthetic α-helical peptides. We examined the formation of a stable uniform ion-selective pore in single-channel electrical recordings. Furthermore, we show that cyclodextrins (CDs) block the peptide pores and determine the kinetics of CD binding and translocation. We suggest that such designed synthetic transmembrane pores will be useful for several applications in biotechnology, including stochastic sensing.


Assuntos
Corynebacterium/metabolismo , Ciclodextrinas/metabolismo , Eletrofisiologia/métodos , Bicamadas Lipídicas/metabolismo , Nanoporos , Fragmentos de Peptídeos/metabolismo , Porinas/metabolismo , Ciclodextrinas/química , Canais Iônicos , Bicamadas Lipídicas/química , Modelos Moleculares , Fragmentos de Peptídeos/química , Porinas/química , Conformação Proteica em alfa-Hélice
20.
Methods Mol Biol ; 2186: 33-48, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32918728

RESUMO

Versatile lipid membrane-inserting nanopores have been made by functionalizing DNA nanostructures with hydrophobic tags. Here, we outline design and considerations to obtain DNA nanopores with the desired dimensions and conductance properties. We further provide guidance on their reconstitution into lipid membranes.


Assuntos
DNA/química , Desenho de Fármacos , Bicamadas Lipídicas/química , Lipídeos de Membrana/química , Nanoporos , Nanoestruturas/química , Nanotecnologia/métodos , Interações Hidrofóbicas e Hidrofílicas
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