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1.
Biophys J ; 104(3): 604-12, 2013 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-23442911

RESUMO

Sphingomyelins (SMs) and ceramides are known to interact favorably in bilayer membranes. Because ceramide lacks a headgroup that could shield its hydrophobic body from unfavorable interactions with water, accommodation of ceramide under the larger phosphocholine headgroup of SM could contribute to their favorable interactions. To elucidate the role of SM headgroup for SM/ceramide interactions, we explored the effects of reducing the size of the phosphocholine headgroup (removing one, two, or three methyls on the choline moiety, or the choline moiety itself). Using differential scanning calorimetry and fluorescence spectroscopy, we found that the size of the SM headgroup had no marked effect on the thermal stability of ordered domains formed by SM analog/palmitoyl ceramide (PCer) interactions. In more complex bilayers composed of a fluid glycerophospholipid, SM analog, and PCer, the thermal stability and molecular order of the laterally segregated gel domains were roughly identical despite variation in SM headgroup size. We suggest that that the association between PCer and SM analogs was stabilized by ceramide's aversion for disordered phospholipids, by interfacial hydrogen bonding between PCer and the SM analogs, and by attractive van der Waals' forces between saturated chains of PCer and SM analogs.


Assuntos
Ceramidas/química , Esfingomielinas/química , Varredura Diferencial de Calorimetria , Colina/química , Ligação de Hidrogênio , Bicamadas Lipídicas/química , Microscopia de Fluorescência , Estrutura Molecular , Temperatura
2.
Biochim Biophys Acta ; 1828(5): 1390-5, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23376330

RESUMO

Sticholysin II (StnII) is a pore-forming toxin from the sea anemone Stichodactyla heliantus which belongs to the large actinoporin family. The toxin binds to sphingomyelin (SM) containing membranes, and shows high binding specificity for this lipid. In this study, we have examined the role of the hydrogen bonding groups of the SM long-chain base (i.e., the 2NH and the 3OH) for StnII recognition. We prepared methylated SM-analogs which had reduced hydrogen bonding capability from 2NH and 3OH. Both surface plasmon resonance experiments, and isothermal titration calorimetry measurements indicated that StnII failed to bind to bilayers containing methylated SM-analogs, whereas clear binding was seen to SM-containing bilayers. StnII also failed to induce calcein release (i.e., pore formation) from vesicles made to contain methylated SM-analogs, but readily induced calcein release from SM-containing vesicles. Molecular modeling of SM docked to the phosphocholine binding site of StnII indicated that the 2NH and 3OH groups were likely to form a hydrogen bond with Tyr135. In addition, it appeared that Tyr111 and Tyr136 could donate hydrogen bonds to phosphate oxygen, thus stabilizing SM binding to the toxin. We conclude that the interfacial hydrogen bonding properties of SM, in addition to the phosphocholine head group, are crucial for high-affinity SM/StnII-interaction.


Assuntos
Venenos de Cnidários/química , Proteínas Citotóxicas Formadoras de Poros/química , Esfingomielinas/química , Lipossomas Unilamelares/química , Animais , Sítios de Ligação , Calorimetria , Venenos de Cnidários/metabolismo , Simulação por Computador , Ligação de Hidrogênio , Cinética , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Estrutura Molecular , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Anêmonas-do-Mar/química , Anêmonas-do-Mar/metabolismo , Esfingomielinas/metabolismo , Ressonância de Plasmônio de Superfície , Lipossomas Unilamelares/metabolismo
3.
Biochim Biophys Acta ; 1818(3): 474-80, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22044798

RESUMO

The aim of this study was to examine how structural properties of different sphingomyelin (SM) analogs affected their substrate properties with sphingomyelinase (SMase) from Bacillus cereus. Using molecular docking and dynamics simulations (for SMase-SM complex), we then attempted to explain the relationship between SM structure and enzyme activity. With both micellar and monolayer substrates, 3O-methylated SM was found not to be degraded by the SMase. 2N-methylated SM was a substrate, but was degraded at about half the rate of its 2NH-SM control. PhytoPSM was readily hydrolyzed by the enzyme. PSM lacking one methyl in the phosphocholine head group was a good substrate, but PSM lacking two or three methyls failed to act as substrates for SMase. Based on literature data, and our docking and MD simulations, we conclude that the 3O-methylated PSM fails to interact with Mg(2+) and Glu53 in the active site, thus preventing hydrolysis. Methylation of 2NH was not crucial for binding to the active site, but appeared to interfere with an induced fit activation of the SMase via interaction with Asp156. An OH on carbon 4 in the long-chain base of phytoPSM appeared not to interfere with the 3OH interacting with Mg(2+) and Glu53 in the active site, and thus did not interfere with catalysis. Removing two or three methyls from the PSM head group apparently increased the positive charge on the terminal N significantly, which most likely led to ionic interactions with Glu250 and Glu155 adjacent to the active site. This likely interaction could have misaligned the SM substrate and hindered proper catalysis.


Assuntos
Bacillus cereus/enzimologia , Proteínas de Bactérias/química , Esfingomielina Fosfodiesterase/química , Esfingomielinas/química , Catálise , Domínio Catalítico , Magnésio/química , Estrutura Molecular , Relação Estrutura-Atividade , Especificidade por Substrato/fisiologia
4.
Biochim Biophys Acta ; 1808(4): 1179-86, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21262197

RESUMO

We have prepared palmitoyl sphingomyelin (PSM) analogs in which either the 2-NH was methylated to NMe, the 3-OH was methylated to OMe, or both were methylated simultaneously. The aim of the study was to determine how such modifications in the membrane interfacial region of the molecules affected interlipid interactions in bilayer membranes. Measuring DPH anisotropy in vesicle membranes prepared from the SM analogs, we observed that methylation decreased gel-phase stability and increased fluid phase disorder, when compared to PSM. Methylation of the 2-NH had the largest effect on gel-phase instability (T(m) was lowered by ~7°C). Atomistic molecular dynamics simulations showed that fluid phase bilayers with methylated SM analogs were more expanded but thinner compared to PSM bilayers. It was further revealed that 3-OH methylation dramatically attenuated hydrogen bonding also via the amide nitrogen, whereas 2-NH methylation did not similarly affect hydrogen bonding via the 3-OH. The interactions of sterols with the methylated SM analogs were markedly affected. 3-OH methylation almost completely eliminated the capacity of the SM analog to form sterol-enriched ordered domains, whereas the 2-NH methylated SM analog formed sterol-enriched domains but these were less thermostable (and thus less ordered) than the domains formed by PSM. Cholestatrienol affinity to bilayers containing methylated SM analogs was also markedly reduced as compared to its affinity for bilayers containing PSM. Molecular dynamics simulations revealed further that cholesterol's bilayer location was deeper in PSM bilayers as compared to the location in bilayers made from methylated SM analogs. This study shows that the interfacial properties of SMs are very important for interlipid interactions and the formation of laterally ordered domains in complex bilayers.


Assuntos
Colesterol/química , Bicamadas Lipídicas/metabolismo , Lipídeos de Membrana/química , Esfingomielinas/química , Anisotropia , Ligação de Hidrogênio , Cinética , Metilação , Estrutura Molecular , Esfingomielinas/metabolismo , Esteróis/química , Temperatura
5.
Biophys J ; 101(12): 2948-56, 2011 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-22208193

RESUMO

To study the role of the interfacial properties of ceramides in their interlipid interactions, we synthesized palmitoylceramide (PCer) analogs in which a methyl group was introduced to the amide-nitrogen or the C3-oxygen of the sphingosine backbone. A differential scanning calorimetry analysis of equimolar mixtures of palmitoylsphingomyelin (PSM) and PCer showed that these sphingolipids formed a complex gel phase that melted between 67°C and 74°C. The PCer analogs also formed gel phases with PSM, but they melted at lower temperatures compared with the system with PCer. In complex bilayers composed of an unsaturated glycerophospholipid, PSM, and cholesterol, the 3O-methylated ceramide formed a cholesterol-poor ordered phase with PSM. However, the 2N-methylated and doubly methylated (2N and 3O) PCer analogs failed to displace sterol from interactions with PSM. Like PCer, the analogs reduced sterol affinity for the complex bilayers, but this effect was most pronounced for the 3O-methylated ceramide. Taken together, our results show that 2N-methylation weakened the ceramide-PSM interactions, whereas the 3O-methylated ceramide behaved more like PCer in interactions with PSM. Our findings are compatible with the view that interlipid interactions between the amide-nitrogen and neighboring lipids are important for the cohesive properties of sphingolipids in membranes, and this also appears to be a valid model for ceramide.


Assuntos
Ceramidas/química , Bicamadas Lipídicas/química , Modelos Químicos , Nitrogênio/química , Oxigênio/química , Esfingosina/química , Metilação
6.
Biophys J ; 99(10): 3300-8, 2010 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-21081078

RESUMO

Sphingomyelins (SMs) and sterols are important constituents of the plasma membrane and have also been identified as major lipid components in membrane rafts. Using SM analogs with decreasing headgroup methylation, we systemically analyzed the effect of headgroup size on membrane properties and interactions with cholesterol. An increase in headgroup size resulted in a decrease in the main phase transition. Atom-scale molecular-dynamics simulations were in agreement with the fluorescence anisotropy experiments, showing that molecular areas increased and acyl chain order decreased with increasing headgroup size. Furthermore, the transition temperatures were constantly higher for SM headgroup analogs compared to corresponding phosphatidylcholine headgroup analogs. The sterol affinity for phospholipid bilayers was assessed using a sterol-partitioning assay and an increased headgroup size increased sterol affinity for the bilayer, with a higher sterol affinity for SM analogs as compared to phosphatidylcholine analogs. Moreover, the size of the headgroup affected the formation and composition of cholesterol-containing ordered domains. Palmitoyl-SM (the largest headgroup) seemed to attract more cholesterol into ordered domains than the other SM analogs with smaller headgroups. The ordering and condensing effect of cholesterol on membrane lipids was also largest for palmitoyl-SM as compared to the smaller SM analogs. The results show that the size of the SM headgroup is crucially important for SM-SM and SM-sterol interactions. Our results further emphasize that interfacial electrostatic interactions are important for stabilizing cholesterol interactions with SMs.


Assuntos
Colesterol/metabolismo , Esfingomielinas/química , Esfingomielinas/metabolismo , Anisotropia , Colestenos/química , Difenilexatrieno/química , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Microdomínios da Membrana/metabolismo , Modelos Biológicos , Transição de Fase , Temperatura de Transição , Lipossomas Unilamelares/química , Água
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