RESUMEN
Steady-state fluorescence anisotropy and dynamic light scattering (DLS) were used to determine the thermotropic properties of lipid systems that act as models for bacterial membranes of Yersinia kristensenii and Proteus mirabilis. Lipid proportions of PE:PG:CL of 0.60:0.20:0.20 and 0.80:0.15:0.05, were used in order to mimic these two membranes respectively. We observed that the introduction of cardiolipin (CL) as a third lipid component of any PE:PG mixture, changes the system's properties considerably. The results obtained by these two techniques show that the main transition temperatures obtained are undoubtedly CL-dependent. Additionally AFM experiments were performed and these results show that even at small concentration CL produces important changes not only in the membrane thermotropic properties, but also in the bilayer structure. In summary, we were able to compare how low and high CL concentration affect bacterial membrane model system properties which can provide a further explanation for the different antibiotic susceptibilities reported for Y. kristensenii and P. mirabilis.
Asunto(s)
Bacterias/química , Cardiolipinas/química , Membrana Celular/química , Fosfatidiletanolaminas/química , Fosfatidilgliceroles/química , Modelos TeóricosRESUMEN
Septic or endotoxic shock is a common cause of death in hospital intensive care units. In the last decade numerous antimicrobial peptides and proteins have been tested in the search for an efficient drug to treat this lethal disease. Now in phase III clinical trials, rBPI(21), a recombinant N-terminal fragment of the bactericidal/permeability-increasing protein (BPI), is a promising drug to reduce lesions caused by meningococcal sepsis. We correlated structural and stability data with functional information of rBPI(21) bound to both model systems of eukaryotic and bacterial membranes. On interaction with membranes, rBPI(21) loses its conformational stability, as studied by circular dichroism. This interaction of rBPI(21) at membrane level was higher in the presence of negatively charged phospholipid relatively to neutral ones, with higher partition coefficients (K(p)), suggesting a preference for bacterial membranes over mammalian membranes. rBPI(21) binding to membranes is reinforced when its disulfide bond is broken due to conformational changes of the protein. This interaction is followed by liposome aggregation due to unfolding, which ensures protein aggregation, and interfacial localization of rBPI(21) in membranes, as studied by extensive quenching by acrylamide and 5-deoxylstearic acid and not by 16-deoxylstearic acid. An uncommon model of the selectivity and mechanism of action is proposed, where membrane induces unfolding of the antimicrobial protein, rBPI(21). The unfolding ensures protein aggregation, established by protein-protein interaction at membrane surface or between adjacent membranes covered by the unfolded protein. This protein aggregation step may lead to membrane perturbation.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Proteínas Sanguíneas/química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Pliegue de Proteína , Proteínas Recombinantes/química , Membrana Celular/química , Membrana Celular/metabolismo , Dicroismo Circular , Disulfuros/química , Luz , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Lipopolisacáridos/metabolismo , Modelos Moleculares , Fosfolípidos/metabolismo , Fosfolípidos/farmacología , Conformación Proteica/efectos de los fármacos , Desnaturalización Proteica , Estabilidad Proteica/efectos de los fármacos , Dispersión de Radiación , Espectrometría de Fluorescencia , Especificidad por Sustrato , Temperatura , Agua/farmacologíaRESUMEN
In Saccharomyces cerevisiae, the diffusion rate of hydrogen peroxide (H2O2) through the plasma membrane decreases during adaptation to H2O2 by means of a mechanism that is still unknown. Here, evidence is presented that during adaptation to H2O2 the anisotropy of the plasma membrane increases. Adaptation to H2O2 was studied at several times (15min up to 90min) by applying the steady-state H2O2 delivery model. For wild-type cells, the steady-state fluorescence anisotropy increased after 30min, or 60min, when using 2-(9-anthroyloxy) stearic acid (2-AS), or diphenylhexatriene (DPH) membrane probe, respectively. Moreover, a 40% decrease in plasma membrane permeability to H2O2 was observed at 15min with a concomitant two-fold increase in catalase activity. Disruption of the ergosterol pathway, by knocking out either ERG3 or ERG6, prevents the changes in anisotropy during H2O2 adaptation. H2O2 diffusion through the plasma membrane in S. cerevisiae cells is not mediated by aquaporins since the H2O2 permeability constant is not altered in the presence of the aquaporin inhibitor mercuric chloride. Altogether, these results indicate that the regulation of the plasma membrane permeability towards H2O2 is mediated by modulation of the biophysical properties of the plasma membrane.
Asunto(s)
Permeabilidad de la Membrana Celular/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/efectos de los fármacos , Adaptación Fisiológica/efectos de los fármacos , Anisotropía , Acuaporinas/metabolismo , Transporte Biológico/efectos de los fármacos , Fenómenos Biofísicos , Biofisica , Fluidez de la Membrana/efectos de los fármacos , Mutación/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
A major requirement to perform structural studies with membrane proteins is not only to define efficient reconstitution protocols, that assure a high incorporation degree in preformed liposomes, but also a protein directionality and topology that mimics its in vivo conditions. For this kind of studies, protein reconstitution in membranes systems via a detergent-mediated pathway is usually successfully adopted, since detergents are generally used in the initial isolation and purification of membrane proteins. In this study we report the reconstitution of OmpF in preformed DMPC and E. coli liposomes using two different techniques for detergent removal: (1) exclusion chromatography and (2) incubation with detergent-adsorbing beads. The incorporation degree was determined by bicinchoninic acid assay and fluorescence anisotropy was used to determine OmpF effect on the structural order of membrane lipids. These results show that protein insertion in membranes depends both on the technique used to remove detergent and on the lipids used to prepare the liposomes. Furthermore, it is possible to state that although the insertion is directly related to the size distributions of proteoliposomes, it could be efficiently recognized by steady-state fluorescence anisotropy. This technique, more popular among cell biologists, can be a very practical and straightforward alternative to DLS to confirm membrane protein insertion.
Asunto(s)
Lípidos/química , Liposomas/química , Proteínas de la Membrana/metabolismo , Membranas/metabolismo , Proteínas/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Cromatografía en Gel/métodos , Detergentes , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Polarización de Fluorescencia/métodos , Luz , Metabolismo de los Lípidos , Liposomas/metabolismo , Lípidos de la Membrana/metabolismo , Proteínas de la Membrana/aislamiento & purificación , Microscopía de Fuerza Atómica/métodos , Proteolípidos , Reproducibilidad de los Resultados , Dispersión de RadiaciónRESUMEN
The analgesic dipeptide kyotorphin (L-Tyr-L-Arg) and an acylated kyotorphin derivative were studied by a combination of theoretical (molecular dynamics simulation and quantum mechanics methods) and experimental (fluorescence and infrared spectroscopies) approaches both in solution and in model systems of membranes. At biological pH the peptides have a neutral net charge. Nevertheless, their phenolic rings interact with phospholipid molecules (partition coefficient varies from 6 x 10(2) to 2 x 10(4), depending on the lipid and pH used) despite being exposed to the aqueous bulk medium. The lowest energy transition dipole moment is displaced from the normal to the lipid bilayer by 20 degrees on average. The observed extensive interaction, pK(a), precise location, and well-defined orientation in membranes combined with the ability to discriminate rigid raftlike membrane domains suggest that kyotorphin meets the structural constraints needed for receptor-ligand interaction. The acylated kyotorphin derivative mimics kyotorphin properties and represents a promising way for entrapment in a drug carrier and transport across the blood-brain barrier.
Asunto(s)
Química Física/métodos , Endorfinas/química , Membrana Dobles de Lípidos/química , Lípidos/química , Barrera Hematoencefálica , Química Farmacéutica/métodos , Portadores de Fármacos , Sistemas de Liberación de Medicamentos , Humanos , Concentración de Iones de Hidrógeno , Iones , Modelos Químicos , Conformación Molecular , Péptidos/química , Teoría CuánticaRESUMEN
Fluorescence techniques were used to study (1) the extent of insertion of the bioactive cyclic dipeptide cyclo(l-tyrosyl-l-prolyl), maculosin, in model systems of membranes of 1, 2-palmitoyl-sn-glycero-3-phosphatidyl choline (DPPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl choline (POPC), (2) its in-depth location in those lipidic membranes, and (3) the influence of cholesterol on the dipeptides's location and orientation. Partition into lipidic bilayers is extensive, mainly for liquid crystalline phase membranes (K(p)=1.3x10(4)). Maculosin locates at the lipid head groups level regardless of the membrane system. Nevertheless, its orientation is lipid phase dependent. When maculosin was inserted in liquid crystalline phase bilayers, its phenolic ring was perpendicular to the membrane surface, whereas it changed orientation when inserted in gel phase membranes. Cholesterol was able to reverse the lipid phase influence on maculosin's orientation.
Asunto(s)
Colesterol/farmacología , Membranas Artificiales , Péptidos Cíclicos/química , Piperazinas/química , Anisotropía , Polarización de Fluorescencia , Espectrometría de FluorescenciaRESUMEN
New drug design has been one of the major challenges to combat bacterial resistance over the past decade. Conventional antibiotics act by destroying bacterial cell wall or by blocking biosynthetic pathways necessary for its survival. Unfortunately, there has been a fast increase in multiresistance, to several conventional antibiotics, in clinical bacterial strains. Previous studies have shown that metalloantibiotics, ternary complexes of antibiotic-metal-phenanthroline, present an increased potential as antimicrobial agents. In this work moxifloxacin, a fourth-generation quinolone, with a broad spectrum of action, and its copper ternary complex (metalloantibiotic) have been study by fluorescence spectroscopy. Partition coefficients were determined and showed that while free moxifloxacin exhibits the same behaviour independently of the lipidic system tested, the metalloantibiotic presents higher partition to liposomes, in a lipid composition-dependent way. These significant differences in the interaction of the metalloantibiotic with model bacteria membranes point out for a putative change in its uptake mechanism with increased drug-lipid interaction potentiating metalloantibiotic influx.
Asunto(s)
Antibacterianos/química , Compuestos Aza/química , Membrana Celular/efectos de los fármacos , Farmacorresistencia Microbiana , Quinolinas/química , Antibacterianos/metabolismo , Compuestos Aza/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Fluoroquinolonas , Liposomas/química , Moxifloxacino , Quinolinas/metabolismo , Espectrometría de FluorescenciaRESUMEN
D-Kyotorphin (D-KTP), the most potent isomer of the endorphin-like dipeptide kyotorphin (KTP), is a good drug candidate for the treatment of chronic pain and is thought to be involved in receptor-mediated processes. According to the "membrane catalysis" model, ligands interact with membrane lipids to attain high local concentrations in the receptor vicinity and to adopt the necessary conformation for docking. Therefore, the interaction and recognition of D-KTP by membranes is potentially important to its increased analgesic effect. In spite of the neutral net charge of D-KTP at pH 7.4, fluorescence spectroscopy reveals that the interaction with large unilamellar vesicles is more extensive than was observed for KTP. The tyrosine residue interacts extensively with rigid membranes, with a location and well-defined orientation in the bilayer. This suggests not only that D-KTP meets the structural constraints needed for receptor-ligand interaction in a manner similar to that of KTP, but also that the stronger membrane interaction and ability to discriminate rigid membrane domains might contribute to its improved analgesic effect.
Asunto(s)
Analgésicos/química , Endorfinas/química , Membrana Dobles de Lípidos/química , Analgésicos/farmacología , Endorfinas/farmacología , Concentración de Iones de Hidrógeno , Membranas/química , Estructura Molecular , Espectrometría de Fluorescencia , Estereoisomerismo , Tirosina/químicaRESUMEN
Neokyotorphin (NKT) is a multifunctional pentapeptide that is involved in biological functions as diverse as analgesia, antihibernatic regulation and proliferation stimulus of tumour cells. The interaction of neokyotorphin with cell membranes is potentially important to all these multiple biological processes since receptor-mediated processes are thought to be involved in neokyotorphin action. Sargent and Schwyzer proposed in their "membrane catalysis" model that ligands interact with membrane lipids in order to adopt the necessary conformation for cell receptors. We have used fluorescence techniques to study the depth, orientation and extent of incorporation of NKT with model membrane systems (lipidic vesicles). The roles of lipid charge, membrane phase and sterol presence were investigated. The phenolic ring of tyrosine is located in a shallow position in membranes. The extent of partition is less in gel crystalline membranes than in liquid crystalline membranes. Addition of cholesterol causes a reorientation of the tyrosine ring at the interface of lipidic bilayers. Lipidic membranes meet all the conditions required for acting as potential "catalysts" in the ligand activity of the multifunctional pentapeptide NKT, because they modulate the exposure and orientation of the phenolic ring, which is most likely involved in docking to receptors.
Asunto(s)
Endorfinas/metabolismo , Membrana Dobles de Lípidos/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/química , Algoritmos , Colesterol/química , Endorfinas/química , Ligandos , Membrana Dobles de Lípidos/química , Liposomas/química , Liposomas/metabolismo , Modelos Moleculares , Péptidos Opioides/química , Péptidos Opioides/metabolismo , Fosfatidilcolinas/química , Fosfatidilgliceroles/química , Unión Proteica , Espectrometría de Fluorescencia , Espectrofotometría Ultravioleta , TermodinámicaRESUMEN
The organization of the polyene antibiotic filipin in membranes containing cholesterol is a controversial matter of debate. Two contradictory models exist, one suggesting a parallel and the other perpendicular organization of filipin with respect to the plane of the membrane. UV-vis linear dichroism, ATR-FTIR, and fluorescence anisotropy decay techniques were combined to study the orientation of filipin in model systems of membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-palmitoyl-sn-glycero-3-phosphocholine (DPPC) with and without cholesterol. Filipin's orientation is determined by the presence/absence of cholesterol when it is inserted in gel crystalline phase model membranes. When cholesterol (33%) is present in DPPC bilayers, filipin stands perpendicular to the membrane surface as expected in "pore-forming" models. At variance, absence of cholesterol leaves filipin in an essentially random organization in the lipidic matrix. In liquid crystalline phase bilayers (POPC) filipin's orientation is perpendicular to the membrane surface even in absence of cholesterol. Thus filipin's activity/organization depends not only on cholesterol presence but also in the lipid phase domain it is inserted in. These findings were combined with spectroscopy and microscopy data in the literature, solving controversial matters of debate.