RESUMO
For highly abundant silica nanomaterials, detrimental effects on proteins and phospholipids are postulated as critical molecular initiating events that involve hydrogen-bonding, hydrophobic, and/or hydrophilic interactions. Here, large unilamellar vesicles with various well-defined phospholipid compositions are used as biomimetic models to recapitulate membranolysis, a process known to be induced by silica nanoparticles in human cells. Differential analysis of the dominant phospholipids determined in membranes of alveolar lung epithelial cells demonstrates that the quaternary ammonium head groups of phosphatidylcholine and sphingomyelin play a critical and dose-dependent role in vesicle binding and rupture by amorphous colloidal silica nanoparticles. Surface modification by either protein adsorption or by covalent coupling of carboxyl groups suppresses the disintegration of these lipid vesicles, as well as membranolysis in human A549 lung epithelial cells by the silica nanoparticles. Furthermore, molecular modeling suggests a preferential affinity of silanol groups for choline head groups, which is also modulated by the pH value. Biomimetic lipid vesicles can thus be used to better understand specific phospholipid-nanoparticle interactions at the molecular level to support the rational design of safe advanced materials.
Assuntos
Nanopartículas , Fosfolipídeos , Humanos , Fosfolipídeos/química , Lipossomas Unilamelares , Dióxido de Silício/química , Colina , Fosfatidilcolinas/química , Lecitinas , Nanopartículas/químicaRESUMO
The integration of functions in materials in order to gain macroscopic effects in response to environmental changes is an ongoing challenge in material science. Here, functions on different hierarchical levels are sequentially linked to translate a pH-triggered conformational transition from the molecular to the macroscopic level to induce directed movements in hydrogels. When the pH is increased, lysine-rich peptide molecules change their conformation into a ß-hairpin structure because of the reduced electrostatic repulsion among the deprotonated amino groups. Coupled to this conformation change is the capability of the ß-hairpin motifs to subsequently assemble into aggregates acting as reversible cross-links, which are used as controlling units to fix a temporary macroscopic shape. A structural function implemented into the hydrogel by a microporous architecture-enabled nondisruptive deformation upon compression by buckling of pore walls and their elastic recovery. Coupled to this structural function is the capability of the porous material to enhance the diffusion of ions into the hydrogel and to keep the dimension of the macroscopic systems almost constant when the additional cross-links are formed or cleaved as it limits the dimensional change of the pore walls. Covalent cross-linking of the hydrogel into a polymer network acted as gear shift to ensure translation of the function on the molecular level to the macroscopic dimension. In this way, the information of a directed shape-shift can be programmed into the material by mechanical deformation and pH-dependent formation of temporary net points. The information could be read out by lowering the pH. The peptides reverted back into their original random coil conformation and the porous polymer network could recover from the previously applied elastic deformation. The level of multifunctionality of the hydrogels can be increased by implementation of additional orthogonal functions such as antimicrobicity by proper selection of multifunctional peptides, which could enable sophisticated biomedical devices.
Assuntos
Hidrogéis/química , Peptídeos/química , Criogéis/química , Difusão , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Polímeros/química , Relação Estrutura-AtividadeRESUMO
Despite extensive use of arginine-rich cell-penetrating peptides (CPPs)-including octaarginine (R8)-as intracellular delivery vectors, mechanisms for their internalization are still under debate. Lipid packing in live cell membranes was characterized using a polarity-sensitive dye (di-4-ANEPPDHQ), and evaluated in terms of generalized polarization. Treatment with membrane curvature-inducing peptides led to significant loosening of the lipid packing, resulting in an enhanced R8 penetration. Pyrenebutyrate (PyB) is known to facilitate R8 membrane translocation by working as a hydrophobic counteranion. Interestingly, PyB also actively induced membrane curvature and perturbed lipid packing. R8 is known to directly cross cell membranes at elevated concentrations. The sites of R8 influx were found to have looser lipid packing than surrounding areas. Lipid packing loosening is proposed as a key factor that governs the membrane translocation of CPPs.
Assuntos
Arginina/metabolismo , Biopolímeros/metabolismo , Peptídeos Penetradores de Células/metabolismo , Lipídeos/química , Sequência de Aminoácidos , Varredura Diferencial de Calorimetria , Membrana Celular/metabolismo , Peptídeos Penetradores de Células/química , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Espectroscopia de Ressonância Magnética , Potenciais da Membrana , Transporte Proteico , Compostos de Piridínio/químicaRESUMO
To understand the molecular mechanisms of amphiphilic membrane-active peptides, one needs to study their interactions with lipid bilayers under ambient conditions. However, it is difficult to control the pH of the sample in biophysical experiments that make use of mechanically aligned multilamellar membrane stacks on solid supports. HPLC-purified peptides tend to be acidic and can change the pH in the sample significantly. Here, we have systematically studied the influence of pH on the lipid interactions of the antimicrobial peptide PGLa embedded in oriented DMPC/DMPG bilayers. Using solid-state NMR (31P, 2H, 19F), both the lipid and peptide components were characterized independently, though in the same oriented samples under typical conditions of maximum hydration. The observed changes in lipid polymorphism were supported by DSC on multilamellar liposome suspensions. On this basis, we can present an optimized sample preparation protocol and discuss the challenges of performing solid-state NMR experiments under controlled pH. DMPC/DMPG bilayers show a significant up-field shift and broadening of the main lipid phase transition temperature when lowering the pH from 10.0 to 2.6. Both, strongly acidic and basic pH, cause a significant degree of lipid hydrolysis, which is exacerbated by the presence of PGLa. The characteristic re-alignment of PGLa from a surface-bound to a tilted state is not affected between pH of 7 to 4 in fluid bilayers. On the other hand, in gel-phase bilayers the peptide remains isotropically mobile under acidic conditions, displays various co-existing orientational states at pH7, and adopts an unknown structural state at basic pH.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Bicamadas Lipídicas/metabolismo , Lipídeos de Membrana/metabolismo , Fosfolipídeos/metabolismo , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/química , Varredura Diferencial de Calorimetria , Dimiristoilfosfatidilcolina/química , Dimiristoilfosfatidilcolina/metabolismo , Concentração de Íons de Hidrogênio , Bicamadas Lipídicas/química , Lipossomos/química , Lipossomos/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Lipídeos de Membrana/química , Dados de Sequência Molecular , Fosfatidilgliceróis/química , Fosfatidilgliceróis/metabolismo , Fosfolipídeos/química , Ligação Proteica , Temperatura de TransiçãoRESUMO
A mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) with the short-chain detergent n-dodecylphosphocholine (DPC) is introduced here as a new membrane-mimetic bicelle system for solid-state NMR structure analysis of membrane proteins in oriented samples. Magnetically aligned DMPC/DPC bicelles are stable over a range of concentrations, with an optimum lipid ratio of q=3:1, and they can be flipped with lanthanide ions. The advantage of DMPC/DPC over established bicelle systems lies in the possibility to use one and the same detergent for purification and NMR analysis of the membrane protein, without any need for detergent exchange. Furthermore, the same batch of protein can be studied in both micelles and bicelles, using liquid-state and solid-state NMR, respectively. The applicability of the DMPC/DPC bicelles is demonstrated here with the (15)N-labeled transmembrane protein TatA.
Assuntos
Dimiristoilfosfatidilcolina/química , Membranas Artificiais , Ressonância Magnética Nuclear Biomolecular/métodos , Fosforilcolina/análogos & derivados , Proteínas de Membrana/química , Fosforilcolina/químicaRESUMO
The bacterial stress-response peptide TisB in Escherichia coli has been suggested to dissipate the transmembrane potential, such that the depletion of ATP levels induces the formation of dormant persister cells which can eventually form biofilms. We studied the structure and membrane interactions of TisB to find out whether it forms pores or other proton-selective channels. Circular dichroism revealed an amphiphilic α-helical structure when reconstituted in lipid vesicles, and oriented circular dichroism showed that the helix assumes a transmembrane alignment. The addition of TisB to dye-loaded vesicles caused leakage only at very high peptide concentration, notably with a Hill coefficient of 2, which suggests that dimers must be involved. Coarse-grained molecular dynamics simulations showed that membrane binding of monomeric TisB is rapid and spontaneous, and transmembrane insertion is energetically feasible. When TisB oligomers are assembled as transmembrane pores, these channels collapse during the simulations, but transmembrane dimers are found to be stable. Given the pattern of charges on the amphiphilic TisB helix, we postulate that antiparallel dimers could be assembled via a ladder of salt bridges. This electrostatic charge-zipper could enable protons to pass along a wire of trapped water molecules across the hydrophobic membrane.
Assuntos
Toxinas Bacterianas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/citologia , Escherichia coli/fisiologia , Bicamadas Lipídicas/metabolismo , Simulação de Dinâmica Molecular , Sequência de Aminoácidos , Toxinas Bacterianas/química , Membrana Celular/química , Membrana Celular/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Bicamadas Lipídicas/química , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Porosidade , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Estresse Fisiológico , Termodinâmica , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismoRESUMO
Being able to control in time and space the positioning, orientation, movement, and sense of rotation of nano- to microscale objects is currently an active research area in nanoscience, having diverse nanotechnological applications. In this paper, we demonstrate unprecedented control and maneuvering of rod-shaped or tubular nanostructures with high aspect ratios which are formed by self-assembling synthetic porphyrins. The self-assembly algorithm, encoded by appended chemical-recognition groups on the periphery of these porphyrins, is the same as the one operating for chlorosomal bacteriochlorophylls (BChl's). Chlorosomes, rod-shaped organelles with relatively long-range molecular order, are the most efficient naturally occurring light-harvesting systems. They are used by green photosynthetic bacteria to trap visible and infrared light of minute intensities even at great depths, e.g., 100 m below water surface or in volcanic vents in the absence of solar radiation. In contrast to most other natural light-harvesting systems, the chlorosomal antennae are devoid of a protein scaffold to orient the BChl's; thus, they are an attractive goal for mimicry by synthetic chemists, who are able to engineer more robust chromophores to self-assemble. Functional devices with environmentally friendly chromophores-which should be able to act as photosensitizers within hybrid solar cells, leading to high photon-to-current conversion efficiencies even under low illumination conditions-have yet to be fabricated. The orderly manner in which the BChl's and their synthetic counterparts self-assemble imparts strong diamagnetic and optical anisotropies and flow/shear characteristics to their nanostructured assemblies, allowing them to be manipulated by electrical, magnetic, or tribomechanical forces.
Assuntos
Complexos de Proteínas Captadores de Luz/síntese química , Porfirinas/síntese química , Anisotropia , Bacterioclorofilas/química , Dicroísmo Circular , Membranas Artificiais , Microscopia Eletrônica de Varredura , Modelos Moleculares , Estrutura MolecularRESUMO
According to their distinct biological functions, membrane-active peptides are generally classified as antimicrobial (AMP), cell-penetrating (CPP), or fusion peptides (FP). The former two classes are known to have some structural and physicochemical similarities, but fusogenic peptides tend to have rather different features and sequences. Nevertheless, we found that many CPPs and some AMPs exhibit a pronounced fusogenic activity, as measured by a lipid mixing assay with vesicles composed of typical eukaryotic lipids. Compared to the HIV fusion peptide (FP23) as a representative standard, all designer-made peptides showed much higher lipid-mixing activities (MSI-103, MAP, transportan, penetratin, Pep1). Native sequences, on the other hand, were less fusogenic (magainin 2, PGLa, gramicidin S), and pre-aggregated ones were inactive (alamethicin, SAP). The peptide structures were characterized by circular dichroism before and after interacting with the lipid vesicles. A striking correlation between the extent of conformational change and the respective fusion activities was found for the series of peptides investigated here. At the same time, the CD data show that lipid mixing can be triggered by any type of conformation acquired upon binding, whether α-helical, ß-stranded, or other. These observations suggest that lipid vesicle fusion can simply be driven by the energy released upon membrane binding, peptide folding, and possibly further aggregation. This comparative study of AMPs, CPPs, and FPs emphasizes the multifunctional aspects of membrane-active peptides, and it suggests that the origin of a peptide (native sequence or designer-made) may be more relevant to define its functional range than any given name.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Penetradores de Células/farmacologia , Fusão de Membrana/efeitos dos fármacos , Dobramento de Proteína , Multimerização Proteica , Lipossomas Unilamelares/metabolismo , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Penetradores de Células/química , Conformação Proteica , Lipossomas Unilamelares/químicaRESUMO
Mechanosensitive channels allow bacteria to respond to osmotic stress by opening a nanometer-sized pore in the cellular membrane. Although the underlying mechanism has been thoroughly studied on the basis of individual channels, the behavior of channel ensembles has yet to be elucidated. This work reveals that mechanosensitive channels of large conductance (MscL) exhibit a tendency to spatially cluster, and demonstrates the functional relevance of clustering. We evaluated the spatial distribution of channels in a lipid bilayer using patch-clamp electrophysiology, fluorescence and atomic force microscopy, and neutron scattering and reflection techniques, coupled with mathematical modeling of the mechanics of a membrane crowded with proteins. The results indicate that MscL forms clusters under a wide range of conditions. MscL is closely packed within each cluster but is still active and mechanosensitive. However, the channel activity is modulated by the presence of neighboring proteins, indicating membrane-mediated protein-protein interactions. Collectively, these results suggest that MscL self-assembly into channel clusters plays an osmoregulatory functional role in the membrane.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli , Canais Iônicos/química , Canais Iônicos/metabolismo , Bicamadas Lipídicas/metabolismo , Membrana Celular/metabolismo , Lipossomos/metabolismo , Microscopia de Força Atômica , Difração de Nêutrons , Ligação Proteica , Espalhamento a Baixo ÂnguloRESUMO
Solid-state (13)C-NMR spectroscopy was used to characterize native cellulose pellicles from two strains of Gluconacetobacter xylinus (ATCC 53582, ATCC 23769), which had been statically cultivated in Hestrin-Schramm (HS) medium containing fully (13)C-labeled beta-D-glucose-U-(13)C(6) as the sole source of carbon. For both samples, the (13)C-NMR chemical shifts were completely assigned for each (13)C-labeled site of cellulose I(alpha) with the aid of 2D refocused INADEQUATE NMR. To determine the principal chemical shift tensor components, a pulse sequence based on the recoupling of anisotropy information (RAI) was applied at 10 kHz MAS. The detailed (13)C tensors of cellulose I(alpha) from different bacterial celluloses are thus available now for the first time, and these results have been compared with previously published data of nonenriched material and with theoretical predictions.
Assuntos
Celulose/química , Gluconacetobacter xylinus/química , Ressonância Magnética Nuclear Biomolecular/métodos , Anisotropia , Bactérias/química , Isótopos de Carbono , Marcação por Isótopo/métodosRESUMO
The level of fatty acid saturation in phospholipids is a crucial determinant of the biophysical properties of the lipid bilayer. Integral membrane proteins are sensitive to changes of their bilayer environment such that their activities and localization can be profoundly affected. When incorporated into phospholipids of mammalian cells, poly-unsaturated fatty acids (PUFAs) determine the mechanical properties of the bilayer thereby affecting several membrane-associated functions such as endo- and exo-cytosis and ion channel/membrane receptor signalling cascades. In order to understand how membrane tension is propagated through poly-unsaturated bilayers, we characterized the effect of lipid saturation on liposome reconstituted MscS and MscL, the two bacterial mechanosensitive ion channels that have for many years served as models of ion- channel-mediated mechanotransduction. The combination of NMR and patch clamp experiments in this study demonstrate that bilayer thinning is the main responsible factor for the modulation of the MscL threshold of activation while a change in transbilayer pressure profile is indicated as the main factor behind the observed modulation of the MscS kinetics. Together, our data offer a novel insight into how the structural shape differences between the two types of mechanosensitive channels determine their differential modulation by poly-unsaturated phospholipids and thus lay the foundation for future functional studies of eukaryotic ion channels involved in the physiology of mechanosensory transduction processes in mammalian cells. SUMMARY: Mechanosensitive channels MscL and MscS are differentially modulated by poly-unsaturated fatty acids in lipid bilayers. MscL becomes sensitized because of increased hydrophobic mismatch while MscS open state is stabilized due to changes in the bilayer lateral pressure profile determined by NMR.
Assuntos
Proteínas de Escherichia coli/metabolismo , Ácidos Graxos Insaturados/metabolismo , Bicamadas Lipídicas/metabolismo , Mecanotransdução Celular/fisiologia , Escherichia coli , Lipossomos/metabolismoRESUMO
Stapling of side chains to stabilize an α-helical structure has been generally associated with an increased uptake of CPPs. Here, we compare four amphiphilic stapled peptides with their linear counterparts in terms of their membrane binding and conformational features in order to correlate these with uptake efficiency and toxicological effects. The impact of lactam stapling was found to vary strongly with regard to the different aspects of peptide-membrane interactions. Nearly all stapled peptides caused less membrane perturbation (vesicle leakage, hemolysis, bacterial lysis) than their linear counterparts. In one case (MAP-1) where stapling enhanced α-helicity in aqueous and lipid environments, leakage was eliminated while cell uptake in HEK293 and HeLa cells remained high, which improved the overall characteristics. The other systems (DRIM, WWSP, KFGF) did not improve, however. The data suggest that cell uptake of amphipathic CPPs correlates with their adopted α-helix content in membranes rather than their helicity in solution.
Assuntos
Antibacterianos/síntese química , Antibacterianos/farmacologia , Membrana Celular/metabolismo , Lactamas/síntese química , Lactamas/farmacologia , Antibacterianos/metabolismo , Bactérias/efeitos dos fármacos , Células HEK293 , Células HeLa , Hemólise/efeitos dos fármacos , Humanos , Lactamas/metabolismo , Membranas Artificiais , Testes de Sensibilidade Microbiana , Modelos Moleculares , Peptídeos/síntese química , Peptídeos/farmacologia , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
An intrinsic clindamycin-resistant Enterococcus faecalis, the most common single species present in teeth after failed root canal therapy, often possesses acquired tetracycline resistance. In these cases, root canal infections are commonly treated with Ledermix(®) paste, which contains demeclocycline, or the new alternative endodontic paste Odontopaste, which contains clindamycin; however, these treatments are often ineffective. We studied the killing activity of the cyclic antimicrobial peptide gramicidin S (GS) against planktonic and biofilm cells of tetracycline-resistant clinical isolates of E. faecalis. The high therapeutic potential of GS for the topical treatment of problematic teeth is based on the rapid bactericidal effect toward the biofilm-forming, tetracycline-resistant E. faecalis. GS reduces the cell number of planktonic cells within 20-40 min at a concentration of 40-80 µg/mL. It kills the cells of pre-grown biofilms at concentrations of 100-200 µg/mL, such that no re-growth is possible. The translocation of the peptide into the cell interior and its complexation with intracellular nucleotides, including the alarmon ppGpp, can explain its anti-biofilm effect. The successful treatment of persistently infected root canals of two volunteers confirms the high effectiveness of GS. The broad GS activity towards resistant, biofilm-forming E. faecalis suggests its applications for approval in root canal medication.
RESUMO
A highly sensitive solid state (19)F-NMR strategy is described to determine the orientation and dynamics of membrane-associated peptides from specific fluorine labels. Several analogues of the antimicrobial peptide PGLa were synthesized with the non-natural amino acid 4-trifluoromethyl-phenylglycine (CF(3)-Phg) at different positions throughout the alpha-helical peptide chain. A simple 1-pulse (19)F experiment allows the simultaneous measurement of both the anisotropic chemical shift and the homonuclear dipolar coupling within the rotating CF(3)-group in a macroscopically oriented membrane sample. The value and sign of the dipolar splitting determines the tilt of the CF(3)-rotational axis, which is rigidly attached to the peptide backbone, with respect to the external magnetic field direction. Using four CF(3)-labeled peptide analogues (with L-CF(3)-Phg at Ile9, Ala10, Ile13, and Ala14) we confirmed that PGLa is aligned at the surface of lipid membranes with its helix axis perpendicular to the bilayer normal at a peptide:lipid ratio of 1:200. We also determined the azimuthal rotation angle of the helix, which agrees well with the orientation expected from its amphiphilic character. Peptide analogues with a D-CF(3)-Phg label resulting from racemization of the amino acid during synthesis were separately collected by HPLC. Their spectra provide additional information about the PGLa structure and orientation but allow only to discriminate qualitatively between multiple solutions. The structural and functional characterization of the individual CF(3)-labeled peptides by circular dichroism and antimicrobial assays showed only small effects for our four substitutions on the hydrophobic face of the helix, but a significant disturbance was observed in a fifth analogue where Ala8 on the hydrophilic face had been replaced. Even though the hydrophobic CF(3)-Phg side chain cannot be utilized in all positions, it allows highly sensitive NMR measurements over a wide range of experimental conditions and dynamic regimes of the peptide.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Dimiristoilfosfatidilcolina/química , Glicina/química , Marcação por Isótopo/métodos , Espectroscopia de Ressonância Magnética/métodos , Fluidez de Membrana , Lipídeos de Membrana/química , Anti-Infecciosos , Radioisótopos de Flúor , Glicina/análogos & derivados , Substâncias Macromoleculares , Membranas Artificiais , Conformação Proteica , Marcadores de SpinRESUMO
Liposomes are used as biocompatible carriers of drugs, peptides, proteins, plasmic DNA, antisense oligonucleotides or ribozymes, for pharmaceutical, cosmetic, and biochemical purposes. The enormous versatility in particle size and in the physical parameters of the lipids affords an attractive potential for constructing tailor-made vehicles for a wide range of applications. Some of the recent literature will be reviewed here and presented from a biophysical point of view, thus providing a background for the more specialized articles in this special issue on liposome technology. Different properties (size, colloidal behavior, phase transitions, and polymorphism) of diverse lipid formulations (liposomes, lipoplexes, cubic phases, emulsions, and solid lipid nanoparticles) for distinct applications (parenteral, transdermal, pulmonary, and oral administration) will be rationalized in terms of common structural, thermodynamic and kinetic parameters of the lipids. This general biophysical basis helps to understand pharmaceutically relevant aspects such as liposome stability during storage and towards serum, the biodistribution and specific targeting of cargo, and how to trigger drug release and membrane fusion. Methods for the preparation and characterization of liposomal formulations in vitro will be outlined, too.
Assuntos
Lipossomos , Animais , Antígenos/administração & dosagem , Fenômenos Biofísicos , Biofísica , Coloides , DNA/administração & dosagem , DNA/química , Estabilidade de Medicamentos , Terapia Genética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas , Lipídeos/química , Lipídeos/farmacocinética , Fluidez de Membrana , Fusão de Membrana , Tamanho da Partícula , Veículos Farmacêuticos , Propriedades de Superfície , Suspensões , TransfecçãoRESUMO
There is an urgent need to coat the surfaces of medical devices, including implants, with antimicrobial agents to reduce the risk of infection. A peptide array technology was modified to permit the screening of short peptides for antimicrobial activity while tethered to a surface. Cellulose-amino-hydroxypropyl ether (CAPE) linker chemistry was used to synthesize, on a cellulose support, peptides that remained covalently bound during biological assays. Among 122 tested sequences, the best surface-tethered 9-, 12-, and 13-mer peptides were found to be highly antimicrobial against bacteria and fungi, as confirmed using alternative surface materials and coupling strategies as well as coupling through the C and N termini of the peptides. Structure-activity modeling of the structural features determining the activity of tethered peptides indicated that the extent and positioning of positive charges and hydrophobic residues were influential in determining activity.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Sequência de Aminoácidos , Antibacterianos/síntese química , Peptídeos Catiônicos Antimicrobianos/síntese química , Celulose/análogos & derivados , Celulose/síntese química , Celulose/química , Avaliação Pré-Clínica de Medicamentos , Interações Hidrofóbicas e Hidrofílicas , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Varredura , Análise Serial de Proteínas , Relação Estrutura-AtividadeRESUMO
It is generally assumed that fusogenic peptides would require a certain conformation, which triggers or participates in the rate-determining step of membrane fusion. Previous structure analyses of the viral fusion peptide from gp41 of HIV-1 have yielded contradictory results, showing either an alpha-helical or a beta-stranded conformation under different conditions. To find out whether either of these conformations is relevant in the actual fusion process, we have placed sterically demanding substitutions into the fusion peptide FP23 to prevent or partially inhibit folding and self-assembly. A single substitution of either D- or L-CF(3)-phenylglycine was introduced in different positions of the sequence, and the capability of these peptide analogues to fuse large unilamellar vesicles was monitored by lipid mixing and dynamic light scattering. If fusion proceeds via a beta-stranded oligomer, then the D- and L-epimers are expected to differ systematically in their activity, since the D-epimers should be unable to form beta-structures due to sterical hindrance. If an alpha-helical conformation is relevant for fusion, then the D-epimers would be slightly disfavoured compared to the L-forms, hence a small systematic difference in fusion activity should be observed. Interestingly, we find that (1) all D- and L-epimers are fusogenically active, though to different extents compared to the wild type, and--most importantly--(ii) there is no systematic preference for either the D- or L-forms. We therefore suggest that a well-structured alpha-helical peptide conformation or a beta-stranded oligomeric assembly can be excluded as the rate-determining state. Instead, fusion appears to involve conformationally disordered peptides with a pronounced structural plasticity.
Assuntos
Fusão de Membrana , Lipídeos de Membrana/química , Modelos Químicos , Lipossomas Unilamelares/química , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/ultraestrutura , Internalização do Vírus , Modelos Moleculares , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
The response of membrane-associated peptides toward the lipid environment or other binding partners can be monitored by solid-state NMR of suitably labeled side chains. Tryptophan is a prominent amino acid in transmembrane helices, and its (19)F-labeled analogues are generally biocompatible and cause little structural perturbation. Hence, we use 5F-Trp as a highly sensitive NMR probe to monitor the conformation and dynamics of the indole ring. To establish this (19)F-NMR strategy, gramicidin A was labeled with 5F-Trp in position 13 or 15, whose chi(1)/chi(2) torsion angles are known from previous (2)H-NMR studies. First, the alignment of the (19)F chemical shift anisotropy tensor within the membrane was deduced by lineshape analysis of oriented samples. Next, the three principal axes of the (19)F chemical shift anisotropy tensor were assigned within the molecular frame of the indole ring. Finally, determination of chi(1)/chi(2) for 5F-Trp in the lipid gel phase showed that the side chain alignment differs by up to 20 degrees from its known conformation in the liquid crystalline state. The sensitivity gain of (19)F-NMR and the reduction in the amount of material was at least 10-fold compared with previous (2)H-NMR studies on the same system and 100-fold compared with (15)N-NMR.