New insight into probe-location dependent polarity and hydration at lipid/water interfaces: comparison between gel- and fluid-phases of lipid bilayers

2016 ◽  
Vol 18 (35) ◽  
pp. 24185-24197 ◽  
Author(s):  
Moirangthem Kiran Singh ◽  
Him Shweta ◽  
Mohammad Firoz Khan ◽  
Sobhan Sen
Keyword(s):  
Lipid Bilayers ◽  
Fluid Phase ◽  
Probe Location ◽  
Fluorescent Molecules ◽  
Insight Into ◽  
Fluid Phases

Location dependent polarity and hydration probed by a new series of 4-aminophthalimide-based fluorescent molecules (4AP-Cn;n= 2–10, 12) show different behaviour at gel- and fluid-phase lipid/water interfaces.

scholarly journals A Förster resonance energy transfer (FRET) approach for enhancing fluorescence contrast in phase-separated membranes

2011 ◽  
Vol 89 (3) ◽  
pp. 423-432
Author(s):  
Daniel M. Carter Ramirez ◽  
Jason Ding ◽  
Jack Guan ◽  
Dusan Vobornik ◽  
Anna Carnini ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Lipid Bilayers ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fluid Phase ◽  
Forster Resonance Energy Transfer ◽  
Förster Resonance Energy Transfer ◽  
Fluorescence Contrast ◽  
Förster Resonance ◽  
Fluid Phases

The partitioning of the dye-labeled lipid probe, NBD-DHPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt)), was examined by fluorescence microscopy in phase-separated lipid bilayers with mixtures of coexisting liquid-ordered and fluid phases. This probe shows slightly higher fluorescence intensity in the ordered domains but undergoes a contrast reversal to give a more strongly fluorescent fluid phase in the presence of >0.2% Texas red-DHPE (TR-DHPE). The change in contrast is shown to result from Förster resonance energy transfer between the NBD donor and TR acceptor in the fluid phase, which has a TR concentration that is approximately 3 times higher than in the domains. An alternate approach using a nitroxide-substituted lipid that partitions into the fluid phase as a quencher, was also examined as a means to enhance the contrast; however, the quencher modified the behaviour of the bilayer. The energy transfer method for enhancing the contrast between ordered and fluid phases was used to examine the morphology of enzyme-treated bilayers.

Direct Measurement of Charge Reversal on Lipid Bilayers using Heterodyne-Detected Second Harmonic Generation Spectroscopy

2019 ◽  
Author(s):  
HanByul Chang ◽  
Paul Ohno ◽  
Yangdongling Liu ◽  
Franz Geiger
Keyword(s):  
Lipid Bilayers ◽  
Surface Potential ◽  
Second Harmonic ◽  
Fluid Phase ◽  
Cationic Polyelectrolyte ◽  
Charge Reversal ◽  
Buried Interfaces ◽  
Phase Transition Temperatures ◽  
Complementary Techniques ◽  
Order Susceptibility

We report the detection of charge reversal induced by the adsorption of a cationic polyelectrolyte, poly(allylamine) hydrochloride (PAH), to buried supported lipid bilayers (SLBs), used as idealized model biological membranes. We observe changes in the surface potential in isolation from other contributors to the total SHG response by extracting the phase-shifted potential-dependent third-order susceptibility from the overall SHG signal. We demonstrate the utility of this technique in detecting both the sign of the surface potential and the point of charge reversal at buried interfaces without any prior information or complementary techniques<i>.</i>Furthermore, isolation of the second-order susceptibility contribution from the overall SHG response allows us to directly monitor changes in the Stern Layer. Finally, we characterize the Stern and Diffuse Layers over single-component SLBs formed from three different zwitterionic lipids of different gel-to-fluid phase transition temperatures (T<sub>m</sub>s). We determine whether the surface potential changes with the physical phase state (gel, transitioning, or fluid) of the SLB and incorporate 20 percent of negatively charged lipids to the zwitterionic SLB to investigate how the surface potential changes with surface charge.

scholarly journals Channel noise in nerve membranes and lipid bilayers

1975 ◽  
Vol 8 (4) ◽  
pp. 451-506 ◽  
Author(s):  
F Conti ◽  
E. Wanke
Keyword(s):  
Brownian Motion ◽  
Lipid Bilayers ◽  
Fluctuation Phenomena ◽  
Channel Noise ◽  
Fluctuation Analysis ◽  
Electronic Charge ◽  
Basic Principles ◽  
Avogadro’S Number ◽  
Insight Into

The basic principles underlying fluctuation phenomena in thermodynamics have long been understood (for reviews see Kubo, 1957; Kubo, Matsuo & Kazuhiro 1973 Lax, 1960). Classical examples of how fluctuation analysis can provide an insight into the corpuscular nature of matter are the determination of Avogadro's number according to Einstein's theory of Brownian motion (see, e.g. Uhlenbeck & Ornstein, 1930; Kac, 1947) and the evaluation of the electronic charge from the shot noise in vacuum tubes (see Van der Ziel, 1970).

scholarly journals Cryo-EM structures of the DCPIB-inhibited volume-regulated anion channel LRRC8A in lipid nanodiscs

2018 ◽  
Author(s):  
David M. Kern ◽  
SeCheol Oh ◽  
Richard K. Hite ◽  
Stephen G. Brohawn
Keyword(s):  
Lipid Bilayers ◽  
Critical Role ◽  
Anion Channel ◽  
Lipid Binding ◽  
Channel Structure ◽  
Anion Channels ◽  
Cryo Electron Microscopy ◽  
Lipid Nanodiscs ◽  
Insight Into

AbstractHypoosmotic conditions activate volume-regulated anion channels in vertebrate cells. These channels are formed by leucine-rich repeat-containing protein 8 (LRRC8) family members and contain LRRC8A in homo- or hetero-hexameric assemblies. Here we present single-particle cryo-electron microscopy structures of LRRC8A in complex with the inhibitor DCPIB reconstituted in lipid nanodiscs. DCPIB plugs the channel like a cork in a bottle - binding in the extracellular selectivity filter and sterically occluding ion conduction. Constricted and expanded structures reveal coupled dilation of cytoplasmic LRRs and the channel pore, suggesting a mechanism for channel gating by internal stimuli. Conformational and symmetry differences between LRRC8A structures determined in detergent micelles and lipid bilayers related to reorganization of intersubunit lipid binding sites demonstrate a critical role for the membrane in determining channel structure. These results provide insight into LRRC8 gating and inhibition and the role of lipids in the structure of an ionic-strength sensing ion channel.

scholarly journals Palmitoylated Cysteines in Chikungunya Virus nsP1 Are Critical for Targeting to Cholesterol-Rich Plasma Membrane Microdomains with Functional Consequences for Viral Genome Replication

2020 ◽  
Vol 94 (10) ◽  
Author(s):  
William Bakhache ◽  
Aymeric Neyret ◽  
Eric Bernard ◽  
Andres Merits ◽  
Laurence Briant
Keyword(s):  
Plasma Membrane ◽  
Lipid Bilayers ◽  
Functional Role ◽  
Membrane Microdomains ◽  
Genome Replication ◽  
Functional Importance ◽  
Late Endosomes ◽  
Plasma Membrane Microdomains ◽  
Insight Into

ABSTRACT In mammalian cells, alphavirus replication complexes are anchored to the plasma membrane. This interaction with lipid bilayers is mediated through the viral methyl/guanylyltransferase nsP1 and reinforced by palmitoylation of cysteine residue(s) in the C-terminal region of this protein. Lipid content of membranes supporting nsP1 anchoring remains poorly studied. Here, we explore the membrane binding capacity of nsP1 with regard to cholesterol. Using the medically important chikungunya virus (CHIKV) as a model, we report that nsP1 cosegregates with cholesterol-rich detergent-resistant membrane microdomains (DRMs), also called lipid rafts. In search for the critical factor for cholesterol partitioning, we identify nsP1 palmitoylated cysteines as major players in this process. In cells infected with CHIKV or transfected with CHIKV trans-replicase plasmids, nsP1, together with the other nonstructural proteins, are detected in DRMs. While the functional importance of CHIKV nsP1 preference for cholesterol-rich membrane domains remains to be determined, we observed that U18666A- and imipramine-induced sequestration of cholesterol in late endosomes redirected nsP1 to these compartments and simultaneously dramatically decreased CHIKV genome replication. A parallel study of Sindbis virus (SINV) revealed that nsP1 from this divergent alphavirus displays a low affinity for cholesterol and only moderately segregates with DRMs. Behaviors of CHIKV and SINV with regard to cholesterol, therefore, match with the previously reported differences in the requirement for nsP1 palmitoylation, which is dispensable for SINV but strictly required for CHIKV replication. Altogether, this study highlights the functional importance of nsP1 segregation with DRMs and provides new insight into the functional role of nsP1 palmitoylated cysteines during alphavirus replication. IMPORTANCE Functional alphavirus replication complexes are anchored to the host cell membranes through the interaction of nsP1 with the lipid bilayers. In this work, we investigate the importance of cholesterol for such an association. We show that nsP1 has affinity for cholesterol-rich membrane microdomains formed at the plasma membrane and identify conserved palmitoylated cysteine(s) in nsP1 as the key determinant for cholesterol affinity. We demonstrate that drug-induced cholesterol sequestration in late endosomes not only redirects nsP1 to this compartment but also dramatically decreases genome replication, suggesting the functional importance of nsP1 targeting to cholesterol-rich plasma membrane microdomains. Finally, we show evidence that nsP1 from chikungunya and Sindbis viruses displays different sensitivity to cholesterol sequestering agents that parallel with their difference in the requirement for nsP1 palmitoylation for replication. This research, therefore, gives new insight into the functional role of palmitoylated cysteines in nsP1 for the assembly of functional alphavirus replication complexes in their mammalian host.

Reconstitution of the complement channel into lipid vesicles and planar bilayers starting from the fluid phase complex

1985 ◽  
Vol 5 (2) ◽  
pp. 129-136 ◽  
Author(s):  
Gianfranco Menestrina ◽  
Flavia Pasquali
Keyword(s):  
Lipid Bilayers ◽  
Membrane Attack Complex ◽  
Fluid Phase ◽  
Lipid Vesicles ◽  
Ionic Channels ◽  
Single Channels ◽  
Planar Bilayers ◽  
Low Concentrations ◽  
Host Membrane ◽  
Phase Complex

Proteolysis of the fluid phase complement complex SC5b-9 transforms it into an arnphiphilic molecule which resembles the membrane attack complex of complement and reconstitutes into lipid vesicles. Complement-containing vesicles prepared in this way can be made to fuse with planar lipid bilayers transferring their protein content to the host membrane. Massive conductance increases can thus be observed, which are due to the insertion of a large number of ionic channels into the membrane. Using low concentrations of vesicles, single channels can be studied.

scholarly journals Structural and Functional Analysis of the Pore-Forming Toxin NetB from Clostridium perfringens

mBio ◽  
2013 ◽  
Vol 4 (1) ◽  
Author(s):  
Xu-Xia Yan ◽  
Corrine J. Porter ◽  
Simon P. Hardy ◽  
David Steer ◽  
A. Ian Smith ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Clostridium Perfringens ◽  
Pore Formation ◽  
Random Mutagenesis ◽  
Planar Lipid Bilayers ◽  
Necrotic Enteritis ◽  
Channel Conductance ◽  
Content Type ◽  
Alpha Hemolysin ◽  
Insight Into

ABSTRACT Clostridium perfringens is an anaerobic bacterium that causes numerous important human and animal diseases, primarily as a result of its ability to produce many different protein toxins. In chickens, C. perfringens causes necrotic enteritis, a disease of economic importance to the worldwide poultry industry. The secreted pore-forming toxin NetB is a key virulence factor in the pathogenesis of avian necrotic enteritis and is similar to alpha-hemolysin, a β-barrel pore-forming toxin from Staphylococcus aureus. To address the molecular mechanisms underlying NetB-mediated tissue damage, we determined the crystal structure of the monomeric form of NetB to 1.8 Å. Structural comparisons with other members of the alpha-hemolysin family revealed significant differences in the conformation of the membrane binding domain. These data suggested that NetB may recognize different membrane receptors or use a different mechanism for membrane-protein interactions. Consistent with this idea, electrophysiological experiments with planar lipid bilayers revealed that NetB formed pores with much larger single-channel conductance than alpha-hemolysin. Channel conductance varied with phospholipid net charge. Furthermore, NetB differed in its ion selectivity, preferring cations over anions. Using hemolysis as a screen, we carried out a random-mutagenesis study that identified several residues that are critical for NetB-induced cell lysis. Mapping of these residues onto the crystal structure revealed that they were clustered in regions predicted to be required for oligomerization or membrane binding. Together these data provide an insight into the mechanism of NetB-mediated pore formation and will contribute to our understanding of the mode of action of this important toxin. IMPORTANCE Necrotic enteritis is an economically important disease of the worldwide poultry industry and is mediated by Clostridium perfringens strains that produce NetB, a β-pore-forming toxin. We carried out structural and functional studies of NetB to provide a mechanistic insight into its mode of action and to assist in the development of a necrotic enteritis vaccine. We determined the structure of the monomeric form of NetB to 1.8 Å, used both site-directed and random mutagenesis to identify key residues that are required for its biological activity, and analyzed pore formation by NetB and its substitution-containing derivatives in planar lipid bilayers.

Molecular Insight into Affinities of Drugs and Their Metabolites to Lipid Bilayers

2013 ◽  
Vol 117 (8) ◽  
pp. 2403-2410 ◽  
Author(s):  
Markéta Paloncýová ◽  
Karel Berka ◽  
Michal Otyepka
Keyword(s):  
Lipid Bilayers ◽  
Insight Into
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