RESUMEN

Bacterial membrane vesicles (MVs) facilitate the long-distance delivery of virulence factors crucial for pathogenicity. The entry and trafficking mechanisms of virulence factors inside host cells are recently emerging; however, whether bacterial MVs can fuse and modulate the physicochemical properties of the host lipid membrane and membrane lipid parameter for fusion remains unknown. In this study, we reconstituted the interaction of bacterial MVs with host cell lipid membranes and quantitatively showed that bacterial MV interaction increases the fluidity, dipole potential, and compressibility of a biologically relevant multicomponent host membrane upon fusion. The presence of cylindrical lipids, such as phosphatidylcholine, and a moderate acyl chain length of C16 help the MV interaction. While significant binding of bacterial MVs to the raft-like lipid membranes with phase-separated regions of the membrane was observed, however, MVs prefer binding to the liquid-disordered regions of the membrane. Furthermore, the elevated levels of cholesterol tend to hinder the interaction of bacterial MVs, as evident from the favorable excess Gibbs free energy of mixing bacterial MVs with host lipid membranes. The findings provide new insights that might have implications for the regulation of host machinery by bacterial pathogens through manipulation of the host membrane properties.

Asunto(s)

RESUMEN

N-terminal residues (770-788) of the S2 glycoprotein of severe acute respiratory syndrome coronavirus (SARS-CoV) have been recognized as a potential fusion peptide that can be involved in the entry of the virus into the host cell. Membrane composition plays an important role in lipid-peptide interaction and the oligomeric status of the peptide. SARS-CoV fusion peptide (S2 fusion peptide) is known to undergo cholesterol-dependent oligomerization in the membrane; however, its significance in membrane fusion is still speculative. This study aimed to investigate the oligomerization of SARS-CoV fusion peptide in a membrane containing phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol, with varying concentrations of cholesterol, and to evaluate peptide-induced membrane fusion to correlate the importance of peptide oligomerization with membrane fusion. Peptide-induced modulation of membrane organization and dynamics was explored by steady-state and time-resolved fluorescence spectroscopic measurements using depth-dependent probes. The results clearly demonstrated the induction of S2 fusion peptide oligomerization by membrane cholesterol and the higher efficiency of the oligomer in promoting membrane fusion compared to its monomeric counterpart. Cholesterol-dependent peptide oligomerization and membrane fusion are important aspects of viral infection since the cholesterol level can change with age as well as with the onset of various pathophysiological conditions.

Asunto(s)

RESUMEN

Membrane fusion is an important event in the life of eukaryotes; occurs in several processes such as endocytosis, exocytosis, cellular trafficking, compartmentalization, import of nutrients and export of waste, vesiculation, inter cellular communication, and fertilization. The enveloped viruses as well utilize fusion between the viral envelope and host cell membrane for infection. The stretch of 20-25 amino acids located at the N-terminus of the fusion protein, known as fusion peptide, plays a decisive role in the fusion process. The stalk model of membrane fusion postulated a common route of bilayer transformation for stalk, transmembrane contact, and pore formation; and fusion peptide is believed to facilitate bilayer transformation to promote membrane fusion. The peptide-induced change in depth-dependent organization and dynamics could provide important information in understanding the role of fusion peptide in membrane fusion. In this review, we have discussed about three depth-dependent properties of the membrane such as rigidity, polarity and heterogeneity, and the impact of fusion peptide on these three membrane properties.

Asunto(s)

RESUMEN

The N-terminal fusion peptide (residues 770-788) of an S2 glycoprotein of the severe acute respiratory syndrome coronavirus (SARS-CoV), exposed upon receptor binding, is crucial for virus entry into the host cell. The fusion peptide alters the membrane organization and dynamics of the host membrane to facilitate membrane fusion. Generally, the effect of the fusion peptide on the membrane is sensitive to the lipid composition of target membranes. In the present work, we have utilized steady-state and time-resolved fluorescence spectroscopy in tandem with circular dichroism spectroscopy to elucidate the binding, oligomeric status, and secondary structure of the fusion peptide and its impact on the depth-dependent membrane organization and dynamics. We have used depth-dependent fluorescence probes, 1,6-diphenyl-1,3,5-hexatriene (DPH) and its trimethylammonium derivative (TMA-DPH), to evaluate the effect of the peptide binding along the bilayer normal. We have exploited the energy transfer efficiency of tryptophan between TMA-DPH and DPH to determine the relative location of the solitary tryptophan present in the membrane-bound fusion peptide. We have further evaluated the effect of membrane cholesterol on the binding and organization of the peptide and the impact of peptide binding on the depth-dependent physical properties of the membrane at various cholesterol concentrations. Our results clearly demonstrate that the membrane cholesterol alters the oligomeric status of the membrane-bound peptide and the effect of peptide binding on the depth-dependent membrane organization and dynamics. The role of cholesterol is important, as the eukaryotic host cells contain a good amount of cholesterol that might be important for the entry of pathogenic viruses.

Asunto(s)

RESUMEN

An envelope glycoprotein, gp41, is crucial for the entry of human immunodeficiency virus (HIV) into the host cell. The 20-23 N-terminal amino acid sequence of gp41 plays an important role in promoting fusion between viral and host cells. Interestingly, the structure and function of the fusion peptide are extremely sensitive to the characteristics of the lipid environment. In this present work, we have extensively utilized steady-state and time-resolved fluorescence spectroscopy in tandem with molecular dynamics simulation to elucidate peptide binding and peptide-induced perturbation to the membrane. We have used two depth-dependent fluorescence probes, 1,6-diphenyl-1,3,5-hexatriene (DPH) and its trimethylammonium derivative (TMA-DPH), to monitor the effect of peptide binding along the bilayer normal and have reconciled the experimental observation with the insights from the simulated molecular events. We have further monitored the effect of membrane cholesterol on peptide-induced membrane perturbation. The molecular dynamics simulation data show that the peptide alters the membrane properties in the vicinity of the peptide and it penetrates to a larger extent into the bilayer when the membrane contains cholesterol. Our results clearly elucidate that cholesterol alters the membrane physical properties in favor of membrane fusion and interaction pattern of the fusion peptide with the membrane in a concentration-dependent fashion. The role of cholesterol is specifically important as the host eukaryotic cells contain a decent amount of cholesterol that might be critical for the entry of HIV into the host cells.

Asunto(s)

RESUMEN

Membrane fusion, one of the most essential processes in the life of eukaryotes, occurs when two separate lipid bilayers merge into a continuous bilayer and internal contents of two separated membranes mingle. There is a certain class of proteins that assist the binding of the viral envelope to the target host cell and catalyzing fusion. All class I viral fusion proteins contain a highly conserved 20-25 amino-acid amphipathic peptide at the N-terminus, which is essential for fusion activity and is termed as the 'fusion peptide'. It has been shown that insertion of fusion peptides into the host membrane and the perturbation in the membrane generated thereby is crucial for membrane fusion. Significant efforts have been given in the last couple of decades to understand the lipid-dependence of structure and function of the fusion peptide in membranes to understand the role of lipid compositions in membrane fusion. In addition, the lipid compositions further change the membrane physical properties and alter the mechanism and extent of membrane fusion. Therefore, lipid compositions modulate membrane fusion by changing membrane physical properties and altering structure of the fusion peptide.

Asunto(s)

RESUMEN

Detection of ions in chemical, biological and environmental samples has gathered tremendous momentum considering the beneficial as well as adverse effects of the ions. Generally, most of the ions are beneficial up to an optimum concentration, beyond which they are toxic to human health. However, most of the fluorescence-based ion sensors are only active in non-aqueous solution because of the low solubility of the sensor molecules in aqueous buffer medium. In the present work, we have demonstrated that encapsulation of an aqueous insoluble thiocarbonohydrazone-locked salicylidene-based macrocyclic ligand in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes allows the selective detection of Zn2+ in aqueous medium with approximately 3-fold enhanced efficiency compared to its efficiency in DMSO medium. We have further modulated the charge of the membrane surface by adding various concentrations of a negatively charged lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), and showed that negative surface charge further enhances the Zn2+ sensing efficiency up to approximately 6-fold. This strategy opens up a new avenue of utilizing organic sensors to detect vital ions in aqueous medium.

RESUMEN

Eugenol is known for its antimicrobial effects against microorganisms responsible for infectious diseases in humans, food-borne pathogens, and oral pathogens. In spite of several reports on the antimicrobial function of eugenol by modulating the structural properties of cell membranes, there is limited information on the influence of eugenol in the lipid membrane. In this work, we explored the effect of eugenol on the organization and dynamics of large unilamellar vesicles (LUVs) of DMPC using the intrinsic fluorescence of eugenol and an extrinsic hydrophobic probe, DPH, in varying phases. The organization and dynamics of the bilayers of DMPC vesicles were monitored by utilizing varieties of steady-state and time-resolved fluorescence measurements. Our results show that eugenol stabilizes the gel phase and elevates the phase-transition temperature of DMPC in a concentration-dependent fashion. Fluorescence lifetime measurements demonstrate that higher eugenol-induced water penetration was observed in fluid-phase membranes. Time-resolved anisotropy measurements demonstrate that eugenol reduces the semiangle of DPH wobbling-in-a-cone in gel-phase membranes, whereas the semiangle remains unaffected in fluid-phase membrane. This implies that the eugenol further orders the gel-phase membrane, and this could be a plausible reason for the eugenol-dependent elevation of the phase-transition temperature of DMPC. We envisage that these results will contribute important information to understand the interaction of eugenol with biological membranes.

Asunto(s)

RESUMEN

Membrane fusion is essential in several cellular processes in the existence of eukaryotic cells such as cellular trafficking, compartmentalization, intercellular communication, sexual reproduction, cell division, and endo- and exocytosis. Membrane fusion proceeds in model membranes as well as biological membranes through the rearrangement of lipids. The stalk hypothesis provides a picture of the general nature of lipid rearrangement based on mechanical properties and phase behavior of water-lipid mesomorphic systems. In spite of extensive research on exploring the mechanism of membrane fusion, a clear molecular understanding of intermediate and pore formation is lacking. In addition, the mechanism by which proteins and peptides reduce the activation energy for stalk and pore formation is not yet clear though there are several propositions on how they catalyze membrane fusion. In this review, we have discussed about various putative functions of fusion peptides by which they reduce activation barrier and thus promote membrane fusion. A careful analysis of the discussed effects of fusion peptides on membranes might open up new possibilities for better understanding of the membrane fusion mechanism.

Asunto(s)

RESUMEN

Intrinsically disordered proteins (IDPs) are under intense analysis due to their structural flexibility and importance in biological functions. Minuscule modulation in the microenvironment induces significant conformational changes in IDPs, and these non-native conformations of the IDPs often induce aggregation and cause cell death. Changes in the membrane composition often change the microenvironment, which promote conformational change and aggregation of IDPs. κ-Casein, an important milk protein, belongs to the class of IDPs containing net negative charges. In this present work, we have studied the interaction of κ-casein with cetyltrimethyl ammonium bromide (CTAB), a positively charged surfactant, utilizing various steady state fluorescence, time-resolved fluorescence and circular dichroism spectroscopy. Our results clearly indicate that κ-casein undergoes at least two conformational transitions in presence of various concentrations of CTAB. The intrinsically disordered κ-casein assumes a partially folded conformation at lower concentration of CTAB, which adopts an unstructured conformation at higher concentration of CTAB. The partially folded conformation of κ-casein at a lower CTAB concentration might be induced by the favorable electrostatic interaction between the positively charged surfactant headgroup and net negative charges of the protein, whereas surfactant nature of CTAB is being pronounced at higher concentration of CTAB.

Asunto(s)

RESUMEN

The fusion peptide of influenza hemagglutinin (HA) is important for cellular entry of influenza virus. Previous studies showed that HA fusion peptide assumes a bent structure in membrane environment, which is extremely important for its fusion-promoting activity. In this work, we have measured the organization and dynamics of the tryptophan (Trp14) residue in presence of sodium dodecyl sulfate (SDS) to investigate the conformational flexibility of tryptophan in membrane-mimetic environment. The steady state and time-resolved fluorescence measurements were employed to investigate the location and rotational flexibility of the tryptophan residue in the SDS micelles. We have calculated the apparent rotational correlation time of Trp14 from the fluorescence anisotropy and fluorescence lifetime data, and apparent rotational correlation time shows a substantial increase in presence of SDS. We have further measured the red edge excitation shift (REES) of Trp14 fluorescence in absence and in presence of two different concentrations of SDS. A large magnitude of REES at post-micellar concentration of SDS indicates the polar confined environment of Trp14 residue in the micellar milieu. Taken together, our results confirm that the tryptophan is located in a motionally restricted interfacial region of micelles. The interfacial location and slow dynamics of Trp14 might be important to adopt the appropriate structure of HA fusion peptide in membrane environment.

Asunto(s)