Visualization of membrane RNAs

PubMed Central

JANAS, TADEUSZ; YARUS, MICHAEL

2003-01-01

Using fluorescence microscopy, we show that previously isolated membrane-binding RNAs coat artificial phospholipid membranes relatively uniformly, except for a frequent tendency to concentrate at bends, membrane junctions, and other unusual sites. Membrane RNAs can also be visualized as single molecules or isolated complexes by atomic force microscopy (AFM) of free RNAs on mica. Finally, RNAs can be seen within membranes by AFM of RNA-liposomes immobilized on hydrophobic mica surfaces. Monomer RNAs appear globular, as expected for small RNAs. When mixed under conditions in which RNAs bind bilayers, RNA 9 and RNA 10 combine to yield about 80% of RNAs as mainly linear oligomers of ≈2–8 molecules. Once inserted in membranes, the RNAs oligomerize further, yielding larger, irregular ropelike structures that prefer the edges of altered lipid patches. These properties can be interpreted in terms of RNA–RNA loop interactions, and the RNA effects on membranes can be explained in terms of an RNA preference for irregular lipid conformations. The RNA-bilayer system poses new opportunities for combining the properties of membranes and RNA in contemporary cells, and also in the ribocytes of an RNA world. PMID:14561885

Agonist properties of a stable hexapeptide analog of neurotensin, N alpha MeArg-Lys-Pro-Trp-tLeu-Leu (NT1).

PubMed

Akunne, H C; Demattos, S B; Whetzel, S Z; Wustrow, D J; Davis, D M; Wise, L D; Cody, W L; Pugsley, T A; Heffner, T G

1995-04-18

The major signal transduction pathway for neurotensin (NT) receptors is the G-protein-dependent stimulation of phospholipase C, leading to the mobilization of intracellular free Ca2+ ([Ca2+]i) and the stimulation of cyclic GMP. We investigated the functional actions of an analog of NT(8-13), N alpha MeArg-Lys-Pro-Trp-tLeu-Leu (NT1), and other NT related analogs by quantitative measurement of the cytosolic free Ca2+ concentration in HT-29 (human colonic adenocarcinoma) cells using the Ca(2+)-sensitive dye fura-2/AM and by effects on cyclic GMP levels in rat cerebellar slices. The NT receptor binding affinities for these analogs to HT-29 cell membranes and newborn (10-day-old) mouse brain membranes were also investigated. Data obtained from HT-29 cell and mouse brain membrane preparations showed saturable single high-affinity sites and binding densities (Bmax) of 130.2 and 87.5 fmol/mg protein, respectively. The respective KD values were 0.47 and 0.39 nM, and the Hill coefficients were 0.99 and 0.92. The low-affinity levocabastine-sensitive site was not present (K1 > 10,000) in either membrane preparation. Although the correlation of binding between HT-29 cell membranes and mouse brain membranes was quite significant (r = 0.92), some of the reference agents had lower binding affinities in the HT-29 cell membranes. The metabolically stable compound NT1 plus other NT analogs and related peptides [NT, NT(8-13), xenopsin, neuromedin N, NT(9-13), kinetensin and (D-Trp11)-NT] increased intracellular Ca2+ levels in HT-29 cells, indicating NT receptor agonist properties. The effect of NT1 in mobilizing [Ca2+]i blocked by SR 48692, a non-peptide NT antagonist. Receptor binding affinities of NT analogs to HT-29 cell membranes were positively correlated with potencies for mobilizing intracellular calcium in the same cells. In addition, NT1 increased cyclic GMP levels in rat cerebellar slices, confirming the latter findings of its NT agonist action. These results substantiate the in vitro NT agonist properties of the hexapeptide NT analog NT1.

Interactions between HIV-1 Neutralizing Antibodies and Model Lipid Membranes imaged with AFM

NASA Astrophysics Data System (ADS)

Zauscher, Stefan; Hardy, Gregory; Alam, Munir; Shapter, Joseph

2012-02-01

Lipid membrane interactions with rare, broadly neutralizing antibodies (NAbs), 2F5 and 4E10, play a critical role in HIV-1 neutralization. Our research is motivated by recent immunization studies that have shown that induction of antibodies that avidly bind the gp41-MPER antigen is not sufficient for neutralization. Rather, it is required that antigen designs induce polyreactive antibodies that recognize MPER antigens as well as the viral lipid membrane. However, the mechanistic details of how membrane properties influence NAb-lipid and NAb-antigen interactions remain unknown. Furthermore, it is well established that the native viral membrane is heterogeneous, representing a mosaic of lipid rafts and protein clustering. However, the size, physical properties, and dynamics of these regions are poorly characterized and their potential roles in HIV-1 neutralization are also unknown. To understand how membrane properties contribute to 2F5/4E10 membrane interactions, we have engineered biomimetic supported lipid bilayers (SLBs) and use atomic force microscopy to visualize membrane domains, antigen clustering, and antibody-membrane interactions at sub-nanometer z-resolution. Our results show that localized binding of HIV-1 antigens and NAbs occur preferentially with the most fluid membrane domain. This supports the theory that NAbs may interact with regions of low lateral lipid forces that allow antibody insertion into the bilayer.

How Lipid Membranes Affect Pore Forming Toxin Activity.

PubMed

Rojko, Nejc; Anderluh, Gregor

2015-12-15

Pore forming toxins (PFTs) evolved to permeate the plasma membrane of target cells. This is achieved in a multistep mechanism that usually involves binding of soluble protein monomer to the lipid membrane, oligomerization at the plane of the membrane, and insertion of part of the polypeptide chain across the lipid membrane to form a conductive channel. Introduced pores allow uncontrolled transport of solutes across the membrane, inflicting damage to the target cell. PFTs are usually studied from the perspective of structure-function relationships, often neglecting the important role of the bulk membrane properties on the PFT mechanism of action. In this Account, we discuss how membrane lateral heterogeneity, thickness, and fluidity influence the pore forming process of PFTs. In general, lipid molecules are more accessible for binding in fluid membranes due to steric reasons. When PFT specifically binds ordered domains, it usually recognizes a specific lipid distribution pattern, like sphingomyelin (SM) clusters or SM/cholesterol complexes, and not individual lipid species. Lipid domains were also suggested to act as an additional concentration platform facilitating PFT oligomerization, but this is yet to be shown. The last stage in PFT action is the insertion of the transmembrane segment across the membranes to build the transmembrane pore walls. Conformational changes are a spontaneous process, and sufficient free energy has to be available for efficient membrane penetration. Therefore, fluid bilayers are permeabilized more readily in comparison to highly ordered and thicker liquid ordered lipid phase (Lo). Energetically more costly insertion into the Lo phase can be driven by the hydrophobic mismatch between the thinner liquid disordered phase (Ld) and large protein complexes, which are unable to tilt like single transmembrane segments. In the case of proteolipid pores, membrane properties can directly modulate pore size, stability, and even selectivity. Finally, events associated with pore formation can modulate properties of the lipid membrane and affect its organization. Model membranes do not necessarily reproduce the physicochemical properties of the native cellular membrane, and caution is needed when transferring results from model to native lipid membranes. In this context, the utilization of novel approaches that enable studying PFTs on living cells at a single molecule level should reveal complex protein-lipid membrane interactions in greater detail.

Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dick, Robert A.; Datta, Siddhartha A. K.; Nanda, Hirsh

2016-05-06

Previously, no retroviral Gag protein has been highly purified in milligram quantities and in a biologically relevant and active form. We have purified Rous sarcoma virus (RSV) Gag protein and in parallel several truncation mutants of Gag and have studied their biophysical properties and membrane interactionsin vitro. RSV Gag is unusual in that it is not naturally myristoylated. From its ability to assemble into virus-like particlesin vitro, we infer that RSV Gag is biologically active. By size exclusion chromatography and small-angle X-ray scattering, Gag in solution appears extended and flexible, in contrast to previous reports on unmyristoylated HIV-1 Gag, whichmore » is compact. However, by neutron reflectometry measurements of RSV Gag bound to a supported bilayer, the protein appears to adopt a more compact, folded-over conformation. At physiological ionic strength, purified Gag binds strongly to liposomes containing acidic lipids. This interaction is stimulated by physiological levels of phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] and by cholesterol. However, unlike HIV-1 Gag, RSV Gag shows no sensitivity to acyl chain saturation. In contrast with full-length RSV Gag, the purified MA domain of Gag binds to liposomes only weakly. Similarly, both an N-terminally truncated version of Gag that is missing the MA domain and a C-terminally truncated version that is missing the NC domain bind only weakly. These results imply that NC contributes to membrane interactionin vitro, either by directly contacting acidic lipids or by promoting Gag multimerization. Retroviruses like HIV assemble at and bud from the plasma membrane of cells. Assembly requires the interaction between thousands of Gag molecules to form a lattice. Previous work indicated that lattice formation at the plasma membrane is influenced by the conformation of monomeric HIV. We have extended this work to the more tractable RSV Gag. Our results show that RSV Gag is highly flexible and can adopt a folded-over conformation on a lipid bilayer, implicating both the N and C termini in membrane binding. In addition, binding of Gag to membranes is diminished when either terminal domain is truncated. RSV Gag membrane association is significantly less sensitive than HIV Gag membrane association to lipid acyl chain saturation. These findings shed light on Gag assembly and membrane binding, critical steps in the viral life cycle and an untapped target for antiretroviral drugs.« less

Novel benzanthrone probes for membrane and protein studies

NASA Astrophysics Data System (ADS)

Ryzhova, Olga; Vus, Kateryna; Trusova, Valeriya; Kirilova, Elena; Kirilov, Georgiy; Gorbenko, Galyna; Kinnunen, Paavo

2016-09-01

The applicability of a series of novel benzanthrone dyes to monitoring the changes in physicochemical properties of lipid bilayer and to differentiating between the native and aggregated protein states has been evaluated. Based on the quantitative parameters of the dye-membrane and dye-protein binding derived from the fluorimetric titration data, the most prospective membrane probes and amyloid tracers have been selected from the group of examined compounds. Analysis of the red edge excitation shifts of the membrane- and amyloid-bound dyes provided information on the properties of benzanthrone binding sites within the lipid and protein matrixes. To understand how amyloid specificity of benzanthrones correlates with their structure, quantitative structure activity relationship (QSAR) analysis was performed involving a range of quantum chemical molecular descriptors. A statistically significant model was obtained for predicting the sensitivity of novel benzanthrone dyes to amyloid fibrils.

Affinity-purified tetanus neurotoxin interaction with synaptic membranes: properties of a protease-sensitive receptor component

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lazarovici, P.; Yavin, E.

1986-11-04

The pharmacokinetic interaction of an affinity-purified /sup 125/I-labeled tetanotoxin fraction with guinea pig brain synaptosomal preparations was investigated. Binding of tetanotoxin was time- and temperature-dependent, was proportional to protein concentration, and was saturable at about 8 x 10/sup -9/ M as estimated by a solid-surface binding assay. Binding was optimal at pH 6.5 under low ionic strength buffer and was almost entirely blocked by gangliosides or antitoxin. In analogy to intact nerve cells, binding of toxin to membranes resulted in a tight association operationally defined as sequestration. Binding and sequestration were abolished after membrane pretreatment with sialidase. The enzyme couldmore » not dissociate the membrane-bound toxin formed at 4 or 37/sup 0/C under low ionic strength conditions, which is in part compatible with internalization as defined in nerve cell cultures. In the latter system the toxin could be removed at 4/sup 0/C but not at 37/sup 0/C. Binding was significantly reduced upon pretreatment of guinea pig brain membranes by a variety of hydrolytic enzymes. It is proposed that, in addition to a ganglioside, interaction of tetanotoxin with synaptic membranes is facilitated by a protein and may also require an appropriate lipid environment. These latter membrane constituents may play a pivotal role in the sequestration of the toxin.« less

Molecular basis of the specific subcellular localization of the C2-like domain of 5-lipoxygenase.

PubMed

Kulkarni, Shilpa; Das, Sudipto; Funk, Colin D; Murray, Diana; Cho, Wonhwa

2002-04-12

The activation of 5-lipoxygenase (5-LO) involves its calcium-dependent translocation to the nuclear envelope, where it catalyzes the two-step transformation of arachidonic acid into leukotriene A(4), leading to the synthesis of various leukotrienes. To understand the mechanism by which 5-LO is specifically targeted to the nuclear envelope, we studied the membrane binding properties of the amino-terminal domain of 5-LO, which has been proposed to have a C2 domain-like structure. The model building, electrostatic potential calculation, and in vitro membrane binding studies of the isolated C2-like domain of 5-LO and selected mutants show that this Ca(2+)-dependent domain selectively binds zwitterionic phosphatidylcholine, which is conferred by tryptophan residues (Trp(13), Trp(75), and Trp(102)) located in the putative Ca(2+)-binding loops. The spatiotemporal dynamics of the enhanced green fluorescence protein-tagged C2-like domain of 5-LO and mutants in living cells also show that the phosphatidylcholine selectivity of the C2-like domain accounts for the specific targeting of 5-LO to the nuclear envelope. Together, these results show that the C2-like domain of 5-LO is a genuine Ca(2+)-dependent membrane-targeting domain and that the subcellular localization of the domain is governed in large part by its membrane binding properties.

Oligomerization but Not Membrane Bending Underlies the Function of Certain F-BAR Proteins in Cell Motility and Cytokinesis.

PubMed

McDonald, Nathan A; Vander Kooi, Craig W; Ohi, Melanie D; Gould, Kathleen L

2015-12-21

F-BAR proteins function in diverse cellular processes by linking membranes to the actin cytoskeleton. Through oligomerization, multiple F-BAR domains can bend membranes into tubules, though the physiological importance of F-BAR-to-F-BAR assemblies is not yet known. Here, we investigate the F-BAR domain of the essential cytokinetic scaffold, Schizosaccharomyces pombe Cdc15, during cytokinesis. Challenging a widely held view that membrane deformation is a fundamental property of F-BARs, we report that the Cdc15 F-BAR binds, but does not deform, membranes in vivo or in vitro, and six human F-BAR domains-including those from Fer and RhoGAP4-share this property. Nevertheless, tip-to-tip interactions between F-BAR dimers are critical for Cdc15 oligomerization and high-avidity membrane binding, stabilization of contractile ring components at the medial cortex, and the fidelity of cytokinesis. F-BAR oligomerization is also critical for Fer and RhoGAP4 physiological function, demonstrating its broad importance to F-BAR proteins that function without membrane bending. Copyright © 2015 Elsevier Inc. All rights reserved.

Novel approach for extraction of quercetin using molecular imprinted membranes

NASA Astrophysics Data System (ADS)

Kamarudin, Siti Fatimah; Ahmad, Mohd Noor; Dzahir, Irfan Hatim Mohamed; Nasir, Azalina Mohamed; Ishak, Noorhidayah; Halim, Nurul Farhanah

2017-12-01

Quercetin imprinted membrane (QIM) was synthesized and applied for the extraction of quercetin. The quercetin imprinted membranes (QIM) were fabricated through a non-covalent approach via surface thermal polymerization. Polyvinylidene fluoride (PVDF) microfiltration membrane was used as a support to improve mechanical stability of the membrane. The thin imprinted layer was formed by copolymerization of acrylamide (AA) as functional monomer and ethylene glycol dimethacrylate (EGDMA) as crosslinker in the presence of quercetin as template in tetrahydrofuran (THF) solution. The Atomic Force Microscopy (AFM) and Field Emission Scanning Electron Microscope (FESEM) were used to visualize the surface of membrane. Batch rebinding and binding kinetic experiments proved that the binding properties of the QIM are higher than non-imprinted membranes (NIM). QIM also have higher selectivity towards quercetin compared to sinensetin and rosmarinic acid.

Comparison of S. cerevisiae F-BAR domain structures reveals a conserved inositol phosphate binding site

PubMed Central

Moravcevic, Katarina; Alvarado, Diego; Schmitz, Karl R.; Kenniston, Jon A.; Mendrola, Jeannine M.; Ferguson, Kathryn M.; Lemmon, Mark A.

2015-01-01

SUMMARY F-BAR domains control membrane interactions in endocytosis, cytokinesis, and cell signaling. Although generally thought to bind curved membranes containing negatively charged phospholipids, numerous functional studies argue that differences in lipid-binding selectivities of F-BAR domains are functionally important. Here, we compare membrane-binding properties of the S. cerevisiae F-BAR domains in vitro and in vivo. Whereas some F-BAR domains (such as Bzz1p and Hof1p F-BARs) bind equally well to all phospholipids, the F-BAR domain from the RhoGAP Rgd1p preferentially binds phosphoinositides. We determined X-ray crystal structures of F-BAR domains from Hof1p and Rgd1p, the latter bound to an inositol phosphate. The structures explain phospholipid-binding selectivity differences, and reveal an F-BAR phosphoinositide binding site that is fully conserved in a mammalian RhoGAP called Gmip, and is partly retained in certain other F-BAR domains. Our findings reveal previously unappreciated determinants of F-BAR domain lipid-binding specificity, and provide a basis for its prediction from sequence. PMID:25620000

Reversible binding kinetics of a cytoskeletal protein at the erythrocyte submembrane.

PubMed Central

Stout, A. L.; Axelrod, D.

1994-01-01

Reversible binding among components of the cellular submembrane cytoskeleton and reversible binding of some of these components with the plasma membrane likely play a role in nonelastic morphological changes and mechanoplastic properties of cells. However, relatively few studies have been devoted to investigating directly the kinetic aspects of the interactions of individual components of the membrane skeleton with the membrane. The experiments described here investigated whether one component of the erythrocyte membrane cytoskeleton, protein 4.1, binds to its sites on the membrane reversibly and if so, whether the different 4.1-binding sites display distinct kinetic behavior. Protein 4.1 is known to stabilize the membrane and to mediate the attachment of spectrin filaments to the membrane. Protein 4.1 previously has been shown to bind to integral membrane proteins band 3, glycophorin C, and to negatively charged phospholipids. To examine the kinetic rates of dissociation of carboxymethyl fluorescein-labeled 4.1 (CF-4.1) to the cytofacial surface of erythrocyte membrane, a special preparation of hemolyzed erythrocyte ghosts was used, in which the ghosts became flattened on a glass surface and exposed their cytofacial surfaces to the solution through a membrane rip in a distinctive characteristic pattern. This preparation was examined by the microscopy technique of total internal reflection/fluorescence recovery after photobleaching (TIR/FRAP). Four different treatments were employed to help identify which membrane binding sites gave rise to the multiplicity of observed kinetic rates. The first treatment, the control, stripped off the native spectrin, actin, 4.1, and ankyrin. About 60% of the CF-4.1 bound to this control binded irreversibly (dissociation time > 20 min), but the remaining approximately 40% binded reversibly with a range of residency times averaging approximately 3 s. The second treatment subjected these stripped membranes to trypsin, which presumably removed most of the band 3. CF-4.1 binded significantly less to these trypsinized membranes and most of the decrease was a loss of the irreversibly binding sites. The third treatment simply preserved the native 4.1 and ankyrin. CF-4.1 binded less to this sample too, and the loss involved both the irreversible and reversible sites. The fourth treatment blocked the gycophorin C sites on the native 4.1-stripped membranes with an antibody. CF-4.1 again binded less to this sample than to a nonimmune serum control, and almost all of the decrease is a loss of irreversible sites. These rest suggest that 1) protein 4.1 binds to membrane or submembrane sites at least in part reversibly ; 2) the most reversible sites are probably not proteinaceous and not glycophorin C, but possibly are phospholipids (especially phosphatidylserine); and 3) TIWRFRAP can successfully examine the fast reversible dynamics of cytoskeletal components binding to biological membranes. Images FIGURE 2 FIGURE 3 FIGURE 4 PMID:7811947

Effect of phloretin on the binding of 1-anilino-8-naphtalene sulfonate (ANS) to 1,2-Dimyristoyl-sn-glycero-3-phosphocoline (DMPC) vesicles in the gel and liquid-crystalline state.

PubMed

Cutró, Andrea C; Montich, Guillermo; Roveri, Oscar A

2015-02-01

Phloretin is a known modifier of the internal dipole potential of lipid membranes. We studied the interaction of phloretin with model lipid membranes and how it influences the membrane dipole organization using ANS as fluorescent probe. The fluorescence increase observed when ANS binds to DMPC liposomes in gel phase (13 °C) was 2.5 times larger in the presence of phloretin. This effect was due to an increase in ANS affinity, which can be related to the known capability of phloretin in decreasing the dipole potential. Conversely, when the experiments were carried out at 33 °C (liquid crystalline phase), phloretin completely inhibited the increase in ANS fluorescence. In addition, phloretin only affected the electrical properties of the membrane in the gel phase, whereas it modifies structural ones in the liquid-crystalline state. We postulate that phloretin was bound only to the DMPC interface in the gel phase decreasing the surface negative charge density without modifying the structural properties of the ANS binding sites. In the liquid-crystalline phase instead, it increased the accessibility of water to the ANS binding sites decreasing the intrinsic affinity and the fluorescence quantum yield of ANS.

The Adipophilin C Terminus Is a Self-folding Membrane-binding Domain That Is Important for Milk Lipid Secretion*

PubMed Central

Chong, Brandi M.; Russell, Tanya D.; Schaack, Jerome; Orlicky, David J.; Reigan, Philip; Ladinsky, Mark; McManaman, James L.

2011-01-01

Cytoplasmic lipid droplets (CLD) in mammary epithelial cells undergo secretion by a unique membrane envelopment process to produce milk lipids. Adipophilin (ADPH/Plin2), a member of the perilipin/PAT family of lipid droplet-associated proteins, is hypothesized to mediate CLD secretion through interactions with apical plasma membrane elements. We found that the secretion of CLD coated by truncated ADPH lacking the C-terminal region encoding a putative four-helix bundle structure was impaired relative to that of CLD coated by full-length ADPH. We used homology modeling and analyses of the solution and membrane binding properties of purified recombinant ADPH C terminus to understand how this region possibly mediates CLD secretion. Homology modeling supports the concept that the ADPH C terminus forms a four-helix bundle motif and suggests that this structure can form stable membrane bilayer interactions. Circular dichroism and protease mapping studies confirmed that the ADPH C terminus is an independently folding α-helical structure that is relatively resistant to urea denaturation. Liposome binding studies showed that the purified C terminus binds to phospholipid membranes through electrostatic dependent interactions, and cell culture studies documented that it localizes to the plasma membrane. Collectively, these data provide direct evidence that the ADPH C terminus forms a stable membrane binding helical structure that is important for CLD secretion. We speculate that interactions between the four-helix bundle of ADPH and membrane phospholipids may be an initial step in milk lipid secretion. PMID:21383012

Spontaneous adsorption of coiled-coil model peptides K and E to a mixed lipid bilayer.

PubMed

Pluhackova, Kristyna; Wassenaar, Tsjerk A; Kirsch, Sonja; Böckmann, Rainer A

2015-03-26

A molecular description of the lipid-protein interactions underlying the adsorption of proteins to membranes is crucial for understanding, for example, the specificity of adsorption or the binding strength of a protein to a bilayer, or for characterizing protein-induced changes of membrane properties. In this paper, we extend an automated in silico assay (DAFT) for binding studies and apply it to characterize the adsorption of the model fusion peptides E and K to a mixed phospholipid/cholesterol membrane using coarse-grained molecular dynamics simulations. In addition, we couple the coarse-grained protocol to reverse transformation to atomistic resolution, thereby allowing to study molecular interactions with high detail. The experimentally observed differential binding of the peptides E and K to membranes, as well as the increased binding affinity of helical over unstructered peptides, could be well reproduced using the polarizable Martini coarse-grained (CG) force field. Binding to neutral membranes is shown to be dominated by initial binding of the positively charged N-terminus to the phospholipid headgroup region, followed by membrane surface-aligned insertion of the peptide at the interface between the hydrophobic core of the membrane and its polar headgroup region. Both coarse-grained and atomistic simulations confirm a before hypothesized snorkeling of lysine side chains for the membrane-bound state of the peptide K. Cholesterol was found to be enriched in peptide vicinity, which is probably of importance for the mechanism of membrane fusion. The applied sequential multiscale method, using coarse-grained simulations for the slow adsorption process of peptides to membranes followed by backward transformation to atomistic detail and subsequent atomistic simulations of the preformed peptide-lipid complexes, is shown to be a versatile approach to study the interactions of peptides or proteins with biomembranes.

Phosphatidylserine lipids and membrane order precisely regulate the activity of Polybia-MP1 peptide.

PubMed

Alvares, Dayane S; Ruggiero Neto, João; Ambroggio, Ernesto E

2017-06-01

Polybia-MP1 (IDWKKLLDAAKQIL-NH 2 ) is a lytic peptide from the Brazilian wasp venom with known anti-cancer properties. Previous evidence indicates that phosphatidylserine (PS) lipids are relevant for the lytic activity of MP1. In agreement with this requirement, phosphatidylserine lipids are translocated to the outer leaflet of cells, and are available for MP1 binding, depending on the presence of liquid-ordered domains. Here, we investigated the effect of PS on MP1 activity when this lipid is reconstituted in membranes of giant or large liposomes with different lipid-phase states. By monitoring the membrane and soluble luminal content of giant unilamellar vesicles (GUVs), using fluorescence confocal microscopy, we were able to determine that MP1 has a pore-forming activity at the membrane level. Liquid-ordered domains, which were phase-separated within the membrane of GUVs, influenced the pore-forming activity of MP1. Experiments evaluating the membrane-binding and lytic activity of MP1 on large unilamellar vesicles (LUVs), with the same lipid composition as GUVs, demonstrated that there was synergy between liquid-ordered domains and PS, which enhanced both activities. Based on our findings, we propose that the physicochemical properties of cancer cell membranes, which possess a much higher concentration of PS than normal cells, renders them susceptible to MP1 binding and lytic pore formation. These results can be correlated with MP1's potent and selective anti-cancer activity and pave the way for future research to develop cancer therapies that harness and exploit the properties of MP1. Copyright © 2017 Elsevier B.V. All rights reserved.

Addition of lysophospholipids with large head groups to cells inhibits Shiga toxin binding.

PubMed

Ailte, Ieva; Lingelem, Anne Berit Dyve; Kavaliauskiene, Simona; Bergan, Jonas; Kvalvaag, Audun Sverre; Myrann, Anne-Grethe; Skotland, Tore; Sandvig, Kirsten

2016-07-26

Shiga toxin (Stx), an AB5 toxin, binds specifically to the neutral glycosphingolipid Gb3 at the cell surface before being transported into cells. We here demonstrate that addition of conical lysophospholipids (LPLs) with large head groups inhibit Stx binding to cells whereas LPLs with small head groups do not. Lysophosphatidylinositol (LPI 18:0), the most efficient LPL with the largest head group, was selected for in-depth investigations to study how the binding of Stx is regulated. We show that the inhibition of Stx binding by LPI is reversible and possibly regulated by cholesterol since addition of methyl-β-cyclodextrin (mβCD) reversed the ability of LPI to inhibit binding. LPI-induced inhibition of Stx binding is independent of signalling and membrane turnover as it occurs in fixed cells as well as after depletion of cellular ATP. Furthermore, data obtained with fluorescent membrane dyes suggest that LPI treatment has a direct effect on plasma membrane lipid packing with shift towards a liquid disordered phase in the outer leaflet, while lysophosphoethanolamine (LPE), which has a small head group, does not. In conclusion, our data show that cellular treatment with conical LPLs with large head groups changes intrinsic properties of the plasma membrane and modulates Stx binding to Gb3.

Gene disruption of dematin causes precipitous loss of erythrocyte membrane stability and severe hemolytic anemia.

PubMed

Lu, Yunzhe; Hanada, Toshihiko; Fujiwara, Yuko; Nwankwo, Jennifer O; Wieschhaus, Adam J; Hartwig, John; Huang, Sha; Han, Jongyoon; Chishti, Athar H

2016-07-07

Dematin is a relatively low abundance actin binding and bundling protein associated with the spectrin-actin junctions of mature erythrocytes. Primary structure of dematin includes a loosely folded core domain and a compact headpiece domain that was originally identified in villin. Dematin's actin binding properties are regulated by phosphorylation of its headpiece domain by cyclic adenosine monophosphate-dependent protein kinase. Here, we used a novel gene disruption strategy to generate the whole body dematin gene knockout mouse model (FLKO). FLKO mice, while born at a normal Mendelian ratio, developed severe anemia and exhibited profound aberrations of erythrocyte morphology and membrane stability. Having no apparent effect on primitive erythropoiesis, FLKO mice show significant enhancement of erythroblast enucleation during definitive erythropoiesis. Using membrane protein analysis, domain mapping, electron microscopy, and dynamic deformability measurements, we investigated the mechanism of membrane instability in FLKO erythrocytes. Although many membrane and cytoskeletal proteins remained at their normal levels, the major peripheral membrane proteins spectrin, adducin, and actin were greatly reduced in FLKO erythrocytes. Our results demonstrate that dematin plays a critical role in maintaining the fundamental properties of the membrane cytoskeleton complex. © 2016 by The American Society of Hematology.

Gene disruption of dematin causes precipitous loss of erythrocyte membrane stability and severe hemolytic anemia

PubMed Central

Lu, Yunzhe; Hanada, Toshihiko; Fujiwara, Yuko; Nwankwo, Jennifer O.; Wieschhaus, Adam J.; Hartwig, John; Huang, Sha; Han, Jongyoon

2016-01-01

Dematin is a relatively low abundance actin binding and bundling protein associated with the spectrin–actin junctions of mature erythrocytes. Primary structure of dematin includes a loosely folded core domain and a compact headpiece domain that was originally identified in villin. Dematin’s actin binding properties are regulated by phosphorylation of its headpiece domain by cyclic adenosine monophosphate–dependent protein kinase. Here, we used a novel gene disruption strategy to generate the whole body dematin gene knockout mouse model (FLKO). FLKO mice, while born at a normal Mendelian ratio, developed severe anemia and exhibited profound aberrations of erythrocyte morphology and membrane stability. Having no apparent effect on primitive erythropoiesis, FLKO mice show significant enhancement of erythroblast enucleation during definitive erythropoiesis. Using membrane protein analysis, domain mapping, electron microscopy, and dynamic deformability measurements, we investigated the mechanism of membrane instability in FLKO erythrocytes. Although many membrane and cytoskeletal proteins remained at their normal levels, the major peripheral membrane proteins spectrin, adducin, and actin were greatly reduced in FLKO erythrocytes. Our results demonstrate that dematin plays a critical role in maintaining the fundamental properties of the membrane cytoskeleton complex. PMID:27073223

A plasma modified cellulose-chitosan porous membrane allows efficient DNA binding and provides antibacterial properties: A step towards developing a new DNA collecting card.

PubMed

Chumwangwapee, Sasiwimon; Chingsungnoen, Artit; Siri, Sineenat

2016-11-01

In forensic DNA analyses, biological specimens are collected and stored for subsequent recovery and analysis of DNA. A cost-effective and efficient DNA recovery approach is therefore a need. This study aims to produce a plasma modified cellulose-chitosan membrane (pCE-CS) that efficiently binds and retains DNA as a potential DNA collecting card. The pCE-CS membrane was produced by a phase separation of ionic liquid dissolving CE and CS in water with subsequent surface-modification by a two-step exposure of argon plasma and nitrogen gas. Through plasma modification, the pCE-CS membrane demonstrated better DNA retention after a washing process and higher rate of DNA recovery as compared with the original CE-CS membrane and the commercial FTA card. In addition, the pCE-CS membrane exhibited anti-bacterial properties against both Escherichia coli and Staphylococcus aureus. The results of this work suggest a potential function of the pCE-CS membrane as a DNA collecting card with a high recovery rate of captured DNA. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Supported Lipid Bilayer/Carbon Nanotube Hybrids

NASA Astrophysics Data System (ADS)

Zhou, Xinjian; Moran-Mirabal, Jose; Craighead, Harold; McEuen, Paul

2007-03-01

We form supported lipid bilayers on single-walled carbon nanotubes and use this hybrid structure to probe the properties of lipid membranes and their functional constituents. We first demonstrate membrane continuity and lipid diffusion over the nanotube. A membrane-bound tetanus toxin protein, on the other hand, sees the nanotube as a diffusion barrier whose strength depends on the diameter of the nanotube. Finally, we present results on the electrical detection of specific binding of streptavidin to biotinylated lipids with nanotube field effect transistors. Possible techniques to extract dynamic information about the protein binding events will also be discussed.

Comparison of Saccharomyces cerevisiae F-BAR domain structures reveals a conserved inositol phosphate binding site.

PubMed

Moravcevic, Katarina; Alvarado, Diego; Schmitz, Karl R; Kenniston, Jon A; Mendrola, Jeannine M; Ferguson, Kathryn M; Lemmon, Mark A

2015-02-03

F-BAR domains control membrane interactions in endocytosis, cytokinesis, and cell signaling. Although they are generally thought to bind curved membranes containing negatively charged phospholipids, numerous functional studies argue that differences in lipid-binding selectivities of F-BAR domains are functionally important. Here, we compare membrane-binding properties of the Saccharomyces cerevisiae F-BAR domains in vitro and in vivo. Whereas some F-BAR domains (such as Bzz1p and Hof1p F-BARs) bind equally well to all phospholipids, the F-BAR domain from the RhoGAP Rgd1p preferentially binds phosphoinositides. We determined X-ray crystal structures of F-BAR domains from Hof1p and Rgd1p, the latter bound to an inositol phosphate. The structures explain phospholipid-binding selectivity differences and reveal an F-BAR phosphoinositide binding site that is fully conserved in a mammalian RhoGAP called Gmip and is partly retained in certain other F-BAR domains. Our findings reveal previously unappreciated determinants of F-BAR domain lipid-binding specificity and provide a basis for its prediction from sequence. Copyright © 2015 Elsevier Ltd. All rights reserved.

Comparison of Saccharomyces cerevisiae F-BAR Domain Structures Reveals a Conserved Inositol Phosphate Binding Site

DOE PAGES

Moravcevic, Katarina; Alvarado, Diego; Schmitz, Karl R.; ...

2015-01-22

F-BAR domains control membrane interactions in endocytosis, cytokinesis, and cell signaling. Although they are generally thought to bind curved membranes containing negatively charged phospholipids, numerous functional studies argue that differences in lipid-binding selectivities of F-BAR domains are functionally important. Here in this paper, we compare membrane-binding properties of the Saccharomyces cerevisiae F-BAR domains in vitro and in vivo. Whereas some F-BAR domains (such as Bzz1p and Hof1p F-BARs) bind equally well to all phospholipids, the F-BAR domain from the RhoGAP Rgd1p preferentially binds phosphoinositides. We determined X-ray crystal structures of F-BAR domains from Hof1p and Rgd1p, the latter bound tomore » an inositol phosphate. The structures explain phospholipid-binding selectivity differences and reveal an F-BAR phosphoinositide binding site that is fully conserved in a mammalian RhoGAP called Gmip and is partly retained in certain other F-BAR domains. In conclusion, our findings reveal previously unappreciated determinants of F-BAR domain lipid-binding specificity and provide a basis for its prediction from sequence.« less

Structural insights into a StART-like domain in Lam4 and its interaction with sterol ligands.

PubMed

Gatta, Alberto T; Sauerwein, Andrea C; Zhuravleva, Anastasia; Levine, Tim P; Matthews, Stephen

2018-01-15

Sterols are essential components of cellular membranes and shape their biophysical properties. The recently discovered family of Lipid transfer proteins Anchored at Membrane contact sites (LAMs) has been suggested to carry out intracellular sterol traffic using StART-like domains. Here, we studied the second StART-like domain of Lam4p from S. cerevisiae by NMR. We show that NMR data are consistent with the StART-like domain structure, and that several functionally important regions within the domain exhibit significant conformational dynamics. NMR titration experiments confirm sterol binding to the canonical sterol-binding site and suggest a role of membrane interactions on the thermodynamics and kinetics of sterol binding. Copyright © 2017 Elsevier Inc. All rights reserved.

Multilayer affinity adsorption of albumin on polymer brushes modified membranes in a continuous-flow system.

PubMed

Hu, Meng-Xin; Li, Xiang; Li, Ji-Nian; Huang, Jing-Jing; Ren, Ge-Rui

2018-02-23

Polymer brushes modified surfaces have been widely used for protein immobilization and isolation. Modification of membranes with polymer brushes increases the surface concentration of affinity ligands used for protein binding. Albumin is one of the transporting proteins and shows a high affinity to bile acids. In this work, the modified membranes with cholic acid-containing polymer brushes can be facilely prepared by the immobilization of cholic acid on the poly(2-hydroxyethyl methacrylate) grafted microporous polypropylene membranes (MPPMs) for affinity adsorption of albumin. ATR/FT-IR and X-ray photoelectron spectroscopy were used to characterize the chemical composition of the modified membranes. Water contact angle measurements were used to analyze the hydrophilic/hydrophobic properties of the membrane surface. The modified MPPMs show a high affinity to albumin and have little non-specific adsorption of hemoglobin. The dynamic binding capacity of albumin in the continous-flow system increases with the cycle number and feed rate as the binding degree of cholic acid is moderate. The highest binding capacity of affinity membranes is about 52.49 g/m 2 membrane, which is about 24 times more than the monolayer binding capacity. These results reveal proteins could be captured in multilayers by the polymer brushes containing affinity ligands similar to the polymer brushes containing ion-exchange groups, which open up the potential of the polymer brushes containing affinity ligands in protein or another components separation. And the cholic acid containing polymer brushes modified membranes has the promising potential for albumin separation and purification rapidly from serum or fermented solution in medical diagnosis and bioseparation. Copyright © 2018 Elsevier B.V. All rights reserved.

Reversible binding of hemoglobin to band 3 constitutes the molecular switch that mediates O2 regulation of erythrocyte properties

PubMed Central

McKenna, Mary M.; Krump, Nathan A.; Zheng, Suilan; Mendelsohn, Laurel; Thein, Swee Lay; Garrett, Lisa J.; Bodine, David M.

2016-01-01

Functional studies have shown that the oxygenation state of the erythrocyte regulates many important pathways, including glucose metabolism, membrane mechanical stability, and cellular adenosine triphosphate (ATP) release. Deoxyhemoglobin (deoxyHb), but not oxyhemoglobin, binds avidly and reversibly to band 3, the major erythrocyte membrane protein. Because band 3 associates with multiple metabolic, solute transport, signal transduction, and structural proteins, the hypothesis naturally arises that the O2-dependent regulation of erythrocyte properties might be mediated by the reversible association of deoxyHb with band 3. To explore whether the band 3–deoxyHb interaction constitutes a “molecular switch” for regulating erythrocyte biology, we have generated transgenic mice with mutations in the deoxyHb-binding domain of band 3. One strain of mouse contains a “humanized” band 3 in which the N-terminal 45 residues of mouse band 3 are replaced by the homologous sequence from human band 3, including the normal human band 3 deoxyHb-binding site. The second mouse contains the same substitution as the first, except the deoxyHb site on band 3 (residues 12-23) has been deleted. Comparison of these animals with wild-type mice demonstrates that the following erythrocyte properties are controlled by the O2-dependent association of hemoglobin with band 3: (1) assembly of a glycolytic enzyme complex on the erythrocyte membrane which is associated with a shift in glucose metabolism between the pentose phosphate pathway and glycolysis, (2) interaction of ankyrin with band 3 and the concomitant regulation of erythrocyte membrane stability, and (3) release of ATP from the red cell which has been linked to vasodilation. PMID:27688804

Effects of membrane properties on the binding activities of the HN and HC heavy-chain domains of botulinum neurotoxin A.

PubMed

Ayyar, B Vijayalakshmi; Atassi, M Zouhair

2016-12-01

Binding behaviors of the H N and the H C domains of BoNT/A were investigated individually to identify if there exist any differences in their interaction with the cell membrane. Recombinant fragments corresponding to both BoNT/A H N and H C regions were prepared (H N 519-845 and H C 967-1296) and their binding to synaptic proteins was verified. The binding behaviors of these heavy-chain domains were analyzed by treating the Neuro 2a, a murine neuroblastoma cell line, with compounds known to alter membrane properties. Cholesterol depletion and lipid raft inhibition increased the binding of H N 519-845 to Neuro 2a cells without affecting H C 967-1296-cell interaction. Sphingolipid depletion decreased the binding of cells to both H C 967-1296 and H N 519-845 whereas, loading exogenous GD1a, on to the Neuro 2a cells, increased the binding of both the peptides to cells. Microtubule disruption of the Neuro 2a cells by nocodazole decreased the binding of both H C 967-1296 and H N 519-845 to the treated cells. Inhibition of the clathrin-mediated endocytosis using dynasore, chlorpromazine or potassium (K + ) depletion buffer lowered the binding of both H C 967-1296 and H N 519-845 to the cells, but seemed to exert a more pronounced effect on the binding of H C 967-1296 than on the binding of H N 519-845. Results indicate that while both the H N and H C domains are involved in the binding of the toxin to neuronal cells there are differences in their behavior which probably stem from their respective amino acid composition and structural location in the toxin three-dimensional structure along with their intended role in translocation and internalization into the cells. Copyright © 2016 Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bidlack, J.M.; Frey, D.K.; Seyed-Mozaffari, A.

The binding properties of 14{beta}-(bromoacetamido)morphine (BAM) and the ability of BAM to irreversibly inhibit opioid binding to rat brain membranes were examined to characterize the affinity and selectivity of BAM as an irreversible affinity ligand for opioid receptors. BAM had the same receptor selectivity as morphine, with a 3-5-fold decrease in affinity for the different types of opioid receptors. When brain membranes were incubated with BAM, followed by extensive washing, opioid binding was restored to control levels. However, when membranes were incubated with dithiothreitol (DTT), followed by BAM, and subsequently washed, 90% of the 0.25 nM ({sup 3}H)(D-Ala{sup 2},(Me)Phe{sup 4},Gly(ol){supmore » 5})enkephalin (DAGO) binding was irreversibly inhibited as a result of the specific alkylation of a sulfhydryl group at the {mu} binding site. This inhibition was dependent on the concentrations of both DTT and BAM. The {mu} receptor specificity of BAM alkylation was demonstrated by the ability of BAM alkylated membranes to still bind the {delta}-selective peptide ({sup 3}H)(D-penicillamine{sup 2},D-penicillamine{sup 5})enkephalin (DPDPE) and (-)-({sup 3}H)bremazocine in the presence of {mu} and {delta} blockers, selective for {kappa} binding sites. Morphine and naloxone partially protected the binding site from alkylation with BAM, while ligands that did not bind to the {mu}s site did not afford protection. These studies have demonstrated that when a disulfide bond at or near {mu} opioid binding sites was reduced, BAM could then alkylate this site, resulting in the specific irreversible labeling of {mu} opioid receptors.« less

Ligand- and drug-binding studies of membrane proteins revealed through circular dichroism spectroscopy.

PubMed

Siligardi, Giuliano; Hussain, Rohanah; Patching, Simon G; Phillips-Jones, Mary K

2014-01-01

A great number of membrane proteins have proven difficult to crystallise for use in X-ray crystallographic structural determination or too complex for NMR structural studies. Circular dichroism (CD) is a fast and relatively easy spectroscopic technique to study protein conformational behaviour. In this review examples of the applications of CD and synchrotron radiation CD (SRCD) to membrane protein ligand binding interaction studies are discussed. The availability of SRCD has been an important advancement in recent progress, most particularly because it can be used to extend the spectral region in the far-UV region (important for increasing the accuracy of secondary structure estimations) and for working with membrane proteins available in only small quantities for which SRCD has facilitated molecular recognition studies. Such studies have been accomplished by probing in the near-UV region the local tertiary structure of aromatic amino acid residues upon addition of chiral or non-chiral ligands using long pathlength cells of small volume capacity. In particular, this review describes the most recent use of the technique in the following areas: to obtain quantitative data on ligand binding (exemplified by the FsrC membrane sensor kinase receptor); to distinguish between functionally similar drugs that exhibit different mechanisms of action towards membrane proteins (exemplified by secretory phospholipase A2); and to identify suitable detergent conditions to observe membrane protein-ligand interactions using stabilised proteins (exemplified by the antiseptic transporter SugE). Finally, the importance of characterising in solution the conformational behaviour and ligand binding properties of proteins in both far- and near-UV regions is discussed. This article is part of a Special Issue entitled: Structural and biophysical characterisation of membrane protein-ligand binding. © 2013.

Structure-function relationship in the binding of snake neurotoxins to the torpedo membrane receptor.

PubMed

Chicheportiche, R; Vincent, J P; Kopeyan, C; Schweitz, H; Lazdunski, M

1975-05-20

The Cys30-Cus34 bridge present in all long neutotoxins (71-74 amino acids, 5 disulfide bridges), but not in short toxins (60-63 amino acids, 4 disulfide bridges), is exposed at the surface since it can be reduced rapidly and selectively by sodium borohydride. Reduction and alkylation of the Cys30-Cys34 bridge of Naja haje neurotoxin III hardly alter the conformational properties of this model long toxin. Although alkylation by iodoacetic acid of th -SH groups liberated by reduction abolishes the toxicity, alkylation by iodoacetamide or ethylenimine does not affect the curarizing efficacy of the toxin. The Cys30-Cys34 bridge is not very important for the toxic activity of long neurotoxins. Reduction of the Cys30-Cys34 bridge followed by alkylation with radioactive iodoacetamide gave a labeled and active toxin which is a convenient derivative for binding experiments to the toxin receptor in membranes of the Torpedo electric organ. The binding capacity of these membrane is 1200 pmol of toxin/mg of membrane protein. The dissociation constant of the modified toxin-receptor complex at pH 7.4, 20 degrees is 10 minus 8m. Reduction with carbroxamidomethylation of the Cys30-Cys34 bridge decreases the affinity of the native Naja haje toxin only by a factor of 15. Carboxymethylation after reduction prevents binding to the membrane receptor. The binding properties of the derivative obtained by reduction and aminoethylation of Cys30-Cys34 are very similar to those of native neurotoxin III; the affinity is decreased only by a factor of 5. Binding properties to Toredo membrane of long neurotoxins (Naja haje neurotoxin III) and short neurotoxins (Naje haje toxin I and Naja mossambica toxin I) have been compared. Dissociation constants of receptor-long neurotoxin and receptor-short neurotoxin complexes are very similar (5.7 minus 8.2 times 10(-10) M at pH 7.4, 20degrees. However, the kinetics of complex formation and complex dissociation are quite different. Short neurotoxins associate 6-7 times faster with the toxin receptor and dissociate about 5-9 times faster that long neurotoxins. Acetylation and dansylation of Lys53 and Lys 27 decrease the affinity of long and short toxins for their receptor by a factor of about 200 at pH 7.4, 20 degrees, mainly because of the slower rate of association with the receptor.

Deciphering the BAR code of membrane modulators.

PubMed

Salzer, Ulrich; Kostan, Julius; Djinović-Carugo, Kristina

2017-07-01

The BAR domain is the eponymous domain of the "BAR-domain protein superfamily", a large and diverse set of mostly multi-domain proteins that play eminent roles at the membrane cytoskeleton interface. BAR domain homodimers are the functional units that peripherally associate with lipid membranes and are involved in membrane sculpting activities. Differences in their intrinsic curvatures and lipid-binding properties account for a large variety in membrane modulating properties. Membrane activities of BAR domains are further modified and regulated by intramolecular or inter-subunit domains, by intermolecular protein interactions, and by posttranslational modifications. Rather than providing detailed cell biological information on single members of this superfamily, this review focuses on biochemical, biophysical, and structural aspects and on recent findings that paradigmatically promote our understanding of processes driven and modulated by BAR domains.

Clostridium botulinum C2 toxin. Identification of the binding site for chloroquine and related compounds and influence of the binding site on properties of the C2II channel.

PubMed

Neumeyer, Tobias; Schiffler, Bettina; Maier, Elke; Lang, Alexander E; Aktories, Klaus; Benz, Roland

2008-02-15

Clostridium botulinum C2 toxin belongs to the family of binary AB type toxins that are structurally organized into distinct enzyme (A, C2I) and binding (B, C2II) components. The proteolytically activated 60-kDa C2II binding component is essential for C2I transport into target cells. It oligomerizes into heptamers and forms channels in lipid bilayer membranes. The C2II channel is cation-selective and can be blocked by chloroquine and related compounds. Residues 303-330 of C2II contain a conserved pattern of alternating hydrophobic and hydrophilic residues, which has been implicated in the formation of two amphipathic beta-strands involved in membrane insertion and channel formation. In the present study, C2II mutants created by substitution of different negatively charged amino acids by alanine-scanning mutagenesis were analyzed in artificial lipid bilayer membranes. The results suggested that most of the C2II mutants formed SDS-resistant oligomers (heptamers) similar to wild type. The mutated negatively charged amino acids did not influence channel properties with the exception of Glu(399) and Asp(426), which are probably localized in the vestibule near the channel entrance. These mutants show a dramatic decrease in their affinity for binding of chloroquine and its analogues. Similarly, F428A, which represents the Phi-clamp in anthrax protective antigen, was mutated in C2II in several other amino acids. The C2II mutants F428A, F428D, F428Y, and F428W not only showed altered chloroquine binding but also had drastically changed single channel properties. The results suggest that amino acids Glu(399), Asp(426), and Phe(428) have a major impact on the function of C2II as a binding protein for C2I delivery into target cells.

Bovine seminal PDC-109 protein: an overview of biochemical and functional properties.

PubMed

Srivastava, N; Jerome, A; Srivastava, S K; Ghosh, S K; Kumar, Amit

2013-04-01

Although long-term storage of bovine semen is desirable for wider use, successful cryopreservation depends on several factors, including various proteins present in seminal plasma. One such group of proteins, viz. bovine seminal plasma (BSP) proteins represents the major protein fraction in bovine seminal plasma. They constitute three major heparin-binding (HB-) acidic proteins secreted by seminal vesicles, viz. BSP-A1/-A2 (PDC-109), BSP-A3 and BSP-30-kDa. By purification studies it was deduced that PDC-109 is a polypeptide of 109 amino acids and contains two tandem repeating fibronectin type-II (Fn-II) domains, preceded by a 23 residue N-terminal domain. Though BSP-A1 and BSP-A2 are biochemically similar they differ only in glycosylation and their mixture is called PDC-109 or gonadostatins. PDC-109 exists as a polydisperse, multimeric self-associated molecule and possesses multifunctional properties, viz. binding to the surface of plasma membrane of spermatozoa causing conformational change in the sperm surface proteins and enhances motility. Besides binding, PDC-109 protein provokes cholesterol efflux from sperm membrane and promotes sperm reservoir by interacting with oviductal membrane. Interaction of sperm with PDC-109 protein induces sperm capacitation and acrosome reaction. However, prolonged exposure of spermatozoa with free floating PDC-109 protein as during processing for preservation, increases cholesterol efflux from spermatozoa. The efflux of sperm membrane cholesterol and disturbance in cholesterol:phospholipids ratio causes destabilization of plasma membrane thereby inducing cryoinjury to the sperm. In this review, the biochemical, functional properties of PDC-109 protein and its role during semen cryopreservation is summarized. Copyright © 2013 Elsevier B.V. All rights reserved.

α-SNAP Interferes with the Zippering of the SNARE Protein Membrane Fusion Machinery

PubMed Central

Park, Yongsoo; Vennekate, Wensi; Yavuz, Halenur; Preobraschenski, Julia; Hernandez, Javier M.; Riedel, Dietmar; Walla, Peter Jomo; Jahn, Reinhard

2014-01-01

Neuronal exocytosis is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Before fusion, SNARE proteins form complexes bridging the membrane followed by assembly toward the C-terminal membrane anchors, thus initiating membrane fusion. After fusion, the SNARE complex is disassembled by the AAA-ATPase N-ethylmaleimide-sensitive factor that requires the cofactor α-SNAP to first bind to the assembled SNARE complex. Using chromaffin granules and liposomes we now show that α-SNAP on its own interferes with the zippering of membrane-anchored SNARE complexes midway through the zippering reaction, arresting SNAREs in a partially assembled trans-complex and preventing fusion. Intriguingly, the interference does not result in an inhibitory effect on synaptic vesicles, suggesting that membrane properties also influence the final outcome of α-SNAP interference with SNARE zippering. We suggest that binding of α-SNAP to the SNARE complex affects the ability of the SNARE complex to harness energy or transmit force to the membrane. PMID:24778182

Biochemical and pharmacological characterization of the thyrotropin releasing hormone (TRH) receptor from clonal GH sub 4 C sub 1 pituitary cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Phillips, W.J.

1987-01-01

The effect of drugs with anesthetic properties on the activity of the pituitary thyrotropin-releasing hormone (TRH) receptor was determined in the clonal GH{sub 4}C{sub 1} somatomammotropic cell line. Classic local anesthetics and other drugs with anesthetic activity inhibited binding of ({sup 3}H)methyl-TRH to cell receptors at concentrations known to produce anesthetic effects on the membrane. The inhibition of TRH receptor binding by tetracaine was competitive and temperature and pH dependent. Verapamil and tetracaine inhibited TRH-stimulated prolactin secretion at concentrations that inhibited peptide binding. TRH-stimulated prolactin secretion was equivalent with or without Ca{sup 2+} channel activity. Verapamil and tetracaine also inhibitedmore » basal prolactin and secretion stimulated by drugs that bypass membrane receptors, db-cAMP and TPA. These results indicate that inhibition of TRH binding and responses by diverse drugs results from an anesthetic effect on the cell membrane.« less

Molecular Dynamics Studies of Structure and Functions of Water-Membrane Interfaces

NASA Technical Reports Server (NTRS)

Pohorille, Andrew; Wilson, Michael A.; DeVincenzi, Donald L. (Technical Monitor)

2001-01-01

A large number of essential cellular processes occur at the interfaces between water and membranes. The selectivity and dynamics of these processes are largely determined by the structural and electrical properties of the water-membrane interface. We investigate these properties by the molecular dynamics method. Over the time scales of the simulations, the membrane undergoes fluctuations described by the capillary wave model. These fluctuations produce occasional thinning defects in the membrane which provide effective pathways for passive transport of ions and small molecules across the membrane. Ions moving through the membrane markedly disrupt its structure and allow for significant water penetration into the membrane interior. Selectivity of transport, with respect to ionic charge, is determined by the interfacial electrostatic potential. Many small molecules. of potential significance in catalysis, bioenergetics and pharmacology, are shown to bind to the interface. The energetics and dynamics of this process will be discussed.

Minimized virus binding for tests of barrier materials.

PubMed Central

Lytle, C D; Routson, L B

1995-01-01

Viruses are used to test the barrier properties of materials. Binding of virus particles during passage through holes in the material may yield misleading test results. The choices of challenge virus and suspending medium may be important for minimizing confounding effects that might arise from such binding. In this study, different surrogate viruses, as well as different support media, were evaluated to determine optimal test parameters. Two membranes with high-binding properties (nitrocellulose and cationic polysulfone) were used as filters to compare binding activities of different surrogate challenge viruses (MS2, phi X174, T7, PRD1, and phi 6) in different media. The media consisted of buffered saline with surfactants, serum, or culture broth as additives. In addition, elution rates of viruses that bound to the membranes were determined. The results suggest that viruses can bind by hydrophobic and electrostatic interactions, with phi X174 displaying the lowest level of binding by either process. The nonionic detergents Triton X-100 and Tween 80 (0.1%) equally minimized hydrophobic interactions. Neither anionic nor cationic surfactants were as effective at nontoxic levels. Serum was effective at reducing both hydrophobic and electrostatic binding, with 2% being sufficient for eliminating binding under our test conditions. Thus, phi X174 remains the best choice as a surrogate virus to test barrier materials, and Triton X-100 (0.1%) remains a good choice for reducing hydrophobic binding. In addition, binding of viruses by barrier materials is unlikely to prevent passage of blood-borne pathogens. PMID:7574603

Mechanisms Underlying the Confined Diffusion of Cholera Toxin B-Subunit in Intact Cell Membranes

PubMed Central

Day, Charles A.; Kenworthy, Anne K.

2012-01-01

Multivalent glycolipid binding toxins such as cholera toxin have the capacity to cluster glycolipids, a process thought to be important for their functional uptake into cells. In contrast to the highly dynamic properties of lipid probes and many lipid-anchored proteins, the B-subunit of cholera toxin (CTxB) diffuses extremely slowly when bound to its glycolipid receptor GM1 in the plasma membrane of living cells. In the current study, we used confocal FRAP to examine the origins of this slow diffusion of the CTxB/GM1 complex at the cell surface, relative to the behavior of a representative GPI-anchored protein, transmembrane protein, and fluorescent lipid analog. We show that the diffusion of CTxB is impeded by actin- and ATP-dependent processes, but is unaffected by caveolae. At physiological temperature, the diffusion of several cell surface markers is unchanged in the presence of CTxB, suggesting that binding of CTxB to membranes does not alter the organization of the plasma membrane in a way that influences the diffusion of other molecules. Furthermore, diffusion of the B-subunit of another glycolipid-binding toxin, Shiga toxin, is significantly faster than that of CTxB, indicating that the confined diffusion of CTxB is not a simple function of its ability to cluster glycolipids. By identifying underlying mechanisms that control CTxB dynamics at the cell surface, these findings help to delineate the fundamental properties of toxin-receptor complexes in intact cell membranes. PMID:22511973

Higher-order assemblies of BAR domain proteins for shaping membranes.

PubMed

Suetsugu, Shiro

2016-06-01

Most cellular organelles contain lipid bilayer membranes. The earliest characterization of cellular organelles was performed by electron microscopy observation of such membranes. However, the precise mechanisms for shaping the membrane in particular subcellular organelles is poorly understood. Classically, the overall cellular shape, i.e. the shape of the plasma membrane, was thought to be governed by the reorganization of cytoskeletal components such as actin and microtubules. The plasma membrane contains various submicron structures such as clathrin-coated pits, caveolae, filopodia and lamellipodia. These subcellular structures are either invaginations or protrusions and are associated with the cytoskeleton. Therefore, it could be hypothesized that there are membrane-binding proteins that cooperates with cytoskeleton in shaping of plasma membrane organelles. Proteins with the Bin-Amphiphysin-Rvs (BAR) domain connect a variety of membrane shapes to actin filaments. The BAR domains themselves bend the membranes by their rigidity and then mold the membranes into tubules through their assembly as spiral polymers, which are thought to be involved in the various submicron structures. Membrane tubulation by polymeric assembly of the BAR domains is supposed to be regulated by binding proteins, binding lipids and the mechanical properties of the membrane. This review gives an overview of BAR protein assembly, describes the significance of the assembly and discusses how to study the assembly in the context of membrane and cellular morphology. The technical problems encountered in microscopic observation of BAR domain assembly are also discussed. © The Author 2016. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Chlorhexidine possesses unique cytotoxic actions in rat thymic lymphocytes: Its relation with electrochemical property of membranes.

PubMed

Nonami, Kayo; Saitoh, Shohei; Nishimura-Danjobara, Yumiko; Ishida, Shiro; Oyama, Yasuo

2016-12-01

Chlorhexidine (CHX) is an antibacterial agent used in various types of pharmaceutical products. Therefore, CHX is easily found around us. Owing to its positive charge, the electrochemical property of cell membranes was assumed to be a key point of cytotoxic action of CHX. Depolarization of membranes attenuated the cytotoxic action of CHX in rat thymic lymphocytes. CHX interfered with annexin V binding to membranes. Manipulations to induce exposure of phosphatidylserine on the outer membrane surface augmented the cytotoxic action of CHX, indicating that changes in the electrochemical property of membranes affected the cytotoxic action of CHX. Hence, CHX might kill cells physiologically undergoing apoptosis, resulting instead in necrotic cell death. However, the threshold CHX concentration in this in vitro study was slightly higher than blood CHX concentrations observed clinically. Therefore, these results may support the safety of CHX use although CHX possesses unique cytotoxic actions described in this study. Copyright © 2016 Elsevier B.V. All rights reserved.

Assembly of streptolysin O pores assessed by quartz crystal microbalance and atomic force microscopy provides evidence for the formation of anchored but incomplete oligomers.

PubMed

Stewart, Sarah E; D'Angelo, Michael E; Paintavigna, Stefania; Tabor, Rico F; Martin, Lisandra L; Bird, Phillip I

2015-01-01

Streptolysin O (SLO) is a bacterial pore forming protein that is part of the cholesterol dependent cytolysin (CDC) family. We have used quartz crystal microbalance with dissipation monitoring (QCM-D) to examine SLO membrane binding and pore formation. In this system, SLO binds tightly to cholesterol-containing membranes, and assembles into partial and complete pores confirmed by atomic force microscopy. SLO binds to the lipid bilayer at a single rate consistent with the Langmuir isotherm model of adsorption. Changes in dissipation illustrate that SLO alters the viscoelastic properties of the bilayer during pore formation, but there is no loss of material from the bilayer as reported for small membrane-penetrating peptides. SLO mutants were used to further dissect the assembly and insertion processes by QCM-D. This shows the signature of SLO in QCM-D changes when pore formation is inhibited, and that bound and inserted SLO forms can be distinguished. Furthermore a pre-pore locked SLO mutant binds reversibly to lipid, suggesting that the partially complete wtSLO forms observed by AFM are anchored to the membrane. Copyright © 2014 Elsevier B.V. All rights reserved.

Characterization, cell-surface expression and ligand-binding properties of different truncated N-terminal extracellular domains of the ionotropic glutamate receptor subunit GluR1.

PubMed

McIlhinney, R A; Molnár, E

1996-04-01

To identify the location of the first transmembrane segment of the GluR1 glutamate receptor subunit artificial stop codons have been introduced into the N-terminal domain at amino acid positions 442, 510, and 563, namely just before and spanning the proposed first two transmembrane regions. The resultant truncated N-terminal fragments of GluR1, termed NT1, NT2, and NT3 respectively were expressed in Cos-7 cells and their cellular distribution and cell-surface expression analysed using an N-terminal antibody to GluR1. All of the fragments were fully glycosylated and were found to be associated with cell membranes but none was secreted. Differential extraction of the cell membranes indicated that both NT1 and NT2 behave as peripheral membrane proteins. In contrast NT3, like the full subunit, has integral membrane protein properties. Furthermore only NT3 is expressed at the cell surface as determined by immunofluorescence and cell-surface biotinylation. Protease protection assays indicated that only NT3 had a cytoplasmic tail. Binding studies using the selective ligand [(3)H]alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate ([(3)H]AMPA) demonstrated that NT3 does not bind ligand. Together these results indicate that the first transmembrane domain of the GluR1 subunit lies between residues 509 and 562, that the N-terminal domain alone cannot form a functional ligand-binding site and that this domain can be targeted to the cell surface provided that it has a transmembrane-spanning region.

Novel interactions of CAPS (Ca2+-dependent activator protein for secretion) with the three neuronal SNARE proteins required for vesicle fusion.

PubMed

Daily, Neil J; Boswell, Kristin L; James, Declan J; Martin, Thomas F J

2010-11-12

CAPS (aka CADPS) is required for optimal vesicle exocytosis in neurons and endocrine cells where it functions to prime the exocytic machinery for Ca(2+)-triggered fusion. Fusion is mediated by trans complexes of the SNARE proteins VAMP-2, syntaxin-1, and SNAP-25 that bridge vesicle and plasma membrane. CAPS promotes SNARE complex formation on liposomes, but the SNARE binding properties of CAPS are unknown. The current work revealed that CAPS exhibits high affinity binding to syntaxin-1 and SNAP-25 and moderate affinity binding to VAMP-2. CAPS binding is specific for a subset of exocytic SNARE protein isoforms and requires membrane integration of the SNARE proteins. SNARE protein binding by CAPS is novel and mediated by interactions with the SNARE motifs in the three proteins. The C-terminal site for CAPS binding on syntaxin-1 does not overlap the Munc18-1 binding site and both proteins can co-reside on membrane-integrated syntaxin-1. As expected for a C-terminal binding site on syntaxin-1, CAPS stimulates SNARE-dependent liposome fusion with N-terminal truncated syntaxin-1 but exhibits impaired activity with C-terminal syntaxin-1 mutants. Overall the results suggest that SNARE complex formation promoted by CAPS may be mediated by direct interactions of CAPS with each of the three SNARE proteins required for vesicle exocytosis.

Novel Interactions of CAPS (Ca2+-dependent Activator Protein for Secretion) with the Three Neuronal SNARE Proteins Required for Vesicle Fusion*

PubMed Central

Daily, Neil J.; Boswell, Kristin L.; James, Declan J.; Martin, Thomas F. J.

2010-01-01

CAPS (aka CADPS) is required for optimal vesicle exocytosis in neurons and endocrine cells where it functions to prime the exocytic machinery for Ca2+-triggered fusion. Fusion is mediated by trans complexes of the SNARE proteins VAMP-2, syntaxin-1, and SNAP-25 that bridge vesicle and plasma membrane. CAPS promotes SNARE complex formation on liposomes, but the SNARE binding properties of CAPS are unknown. The current work revealed that CAPS exhibits high affinity binding to syntaxin-1 and SNAP-25 and moderate affinity binding to VAMP-2. CAPS binding is specific for a subset of exocytic SNARE protein isoforms and requires membrane integration of the SNARE proteins. SNARE protein binding by CAPS is novel and mediated by interactions with the SNARE motifs in the three proteins. The C-terminal site for CAPS binding on syntaxin-1 does not overlap the Munc18-1 binding site and both proteins can co-reside on membrane-integrated syntaxin-1. As expected for a C-terminal binding site on syntaxin-1, CAPS stimulates SNARE-dependent liposome fusion with N-terminal truncated syntaxin-1 but exhibits impaired activity with C-terminal syntaxin-1 mutants. Overall the results suggest that SNARE complex formation promoted by CAPS may be mediated by direct interactions of CAPS with each of the three SNARE proteins required for vesicle exocytosis. PMID:20826818

Investigation of the Lipid Binding Properties of the Marburg Virus Matrix Protein VP40.

PubMed

Wijesinghe, Kaveesha J; Stahelin, Robert V

2015-12-30

Marburg virus (MARV), which belongs to the virus family Filoviridae, causes hemorrhagic fever in humans and nonhuman primates that is often fatal. MARV is a lipid-enveloped virus that during the replication process extracts its lipid coat from the plasma membrane of the host cell it infects. MARV carries seven genes, one of which encodes its matrix protein VP40 (mVP40), which regulates the assembly and budding of the virions. Currently, little information is available on mVP40 lipid binding properties. Here, we have investigated the in vitro and cellular mechanisms by which mVP40 associates with lipid membranes. mVP40 associates with anionic membranes in a nonspecific manner that is dependent upon the anionic charge density of the membrane. These results are consistent with recent structural determination of mVP40, which elucidated an mVP40 dimer with a flat and extensive cationic lipid binding interface. Marburg virus (MARV) is a lipid-enveloped filamentous virus from the family Filoviridae. MARV was discovered in 1967, and yet little is known about how its seven genes are used to assemble and form a new viral particle in the host cell it infects. The MARV matrix protein VP40 (mVP40) underlies the inner leaflet of the virus and regulates budding from the host cell plasma membrane. In vitro and cellular assays in this study investigated the mechanism by which mVP40 associates with lipids. The results demonstrate that mVP40 interactions with lipid vesicles or the inner leaflet of the plasma membrane are electrostatic but nonspecific in nature and are dependent on the anionic charge density of the membrane surface. Small molecules that can disrupt lipid trafficking or reduce the anionic charge of the plasma membrane interface may be useful in inhibiting assembly and budding of MARV. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

The Solution Structure, Binding Properties, and Dynamics of the Bacterial Siderophore-binding Protein FepB*

PubMed Central

Chu, Byron C. H.; Otten, Renee; Krewulak, Karla D.; Mulder, Frans A. A.; Vogel, Hans J.

2014-01-01

The periplasmic binding protein (PBP) FepB plays a key role in transporting the catecholate siderophore ferric enterobactin from the outer to the inner membrane in Gram-negative bacteria. The solution structures of the 34-kDa apo- and holo-FepB from Escherichia coli, solved by NMR, represent the first solution structures determined for the type III class of PBPs. Unlike type I and II PBPs, which undergo large “Venus flytrap” conformational changes upon ligand binding, both forms of FepB maintain similar overall folds; however, binding of the ligand is accompanied by significant loop movements. Reverse methyl cross-saturation experiments corroborated chemical shift perturbation results and uniquely defined the binding pocket for gallium enterobactin (GaEnt). NMR relaxation experiments indicated that a flexible loop (residues 225–250) adopted a more rigid and extended conformation upon ligand binding, which positioned residues for optimal interactions with the ligand and the cytoplasmic membrane ABC transporter (FepCD), respectively. In conclusion, this work highlights the pivotal role that structural dynamics plays in ligand binding and transporter interactions in type III PBPs. PMID:25173704

Analysis of Protein Interactions at Native Chloroplast Membranes by Ellipsometry

PubMed Central

Kriechbaumer, Verena; Nabok, Alexei; Mustafa, Mohd K.; Al-Ammar, Rukaiah; Tsargorodskaya, Anna; Smith, David P.; Abell, Ben M.

2012-01-01

Membrane bound receptors play vital roles in cell signaling, and are the target for many drugs, yet their interactions with ligands are difficult to study by conventional techniques due to the technical difficulty of monitoring these interactions in lipid environments. In particular, the ability to analyse the behaviour of membrane proteins in their native membrane environment is limited. Here, we have developed a quantitative approach to detect specific interactions between low-abundance chaperone receptors within native chloroplast membranes and their soluble chaperone partners. Langmuir-Schaefer film deposition was used to deposit native chloroplasts onto gold-coated glass slides, and interactions between the molecular chaperones Hsp70 and Hsp90 and their receptors in the chloroplast membranes were detected and quantified by total internal reflection ellipsometry (TIRE). We show that native chloroplast membranes deposited on gold-coated glass slides using Langmuir-Schaefer films retain functional receptors capable of binding chaperones with high specificity and affinity. Taking into account the low chaperone receptor abundance in native membranes, these binding properties are consistent with data generated using soluble forms of the chloroplast chaperone receptors, OEP61 and Toc64. Therefore, we conclude that chloroplasts have the capacity to selectively bind chaperones, consistent with the notion that chaperones play an important role in protein targeting to chloroplasts. Importantly, this method of monitoring by TIRE does not require any protein labelling. This novel combination of techniques should be applicable to a wide variety of membranes and membrane protein receptors, thus presenting the opportunity to quantify protein interactions involved in fundamental cellular processes, and to screen for drugs that target membrane proteins. PMID:22479632

A versatile method for fabricating ion-exchange hydrogel nanofibrous membranes with superb biomolecule adsorption and separation properties.

PubMed

Lv, Huan; Wang, Xueqin; Fu, Qiuxia; Si, Yang; Yin, Xia; Li, Xiaoran; Sun, Gang; Yu, Jianyong; Ding, Bin

2017-11-15

Construction ion-exchange membranes with superb biomolecules adsorption and purification performance plays a greatly important role in the fields of biotechnological and biopharmaceutical industry, yet still remains an extremely challenging. Herein, we in situ synthesized the cis-butenedioic anhydride grafted poly(vinyl alcohol) hydrogel nanofibrous membranes (CBA-g-PVA HNFM) by combining electrospinning technique with the grafting-copolymerization crosslinking. Taking advantages of the large specific surface area which could provide numerous sites available for functional groups and biomolecules binding, highly tortuous and interconnected porous channel for biomolecules transfer, and enhanced mechanical strength, the resultant CBA-g-PVA HNFM exhibited relatively high binding amount of 170mgg -1 , rapid equilibrium time of 8h towards the biomolecule template of lysozyme, and the performance could be tailored by regulating the buffer properties and protein concentrations. Additionally, the resultant functional HNFM also possessed superior acid resistance property, excellent reversibility and regeneration performance. More importantly, the obtained CBA-g-PVA HNFM could directly extract lysozyme from fresh chicken eggs with capacity of 125mgg -1 , exhibiting excellent practical application properties. The fabrication of proposed CBA-g-PVA HNFM offers a feasible alternative for construction of ion-exchange chromatograph column for bio-separation and purification engineering. Copyright © 2017 Elsevier Inc. All rights reserved.

α-SNAP interferes with the zippering of the SNARE protein membrane fusion machinery.

PubMed

Park, Yongsoo; Vennekate, Wensi; Yavuz, Halenur; Preobraschenski, Julia; Hernandez, Javier M; Riedel, Dietmar; Walla, Peter Jomo; Jahn, Reinhard

2014-06-06

Neuronal exocytosis is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Before fusion, SNARE proteins form complexes bridging the membrane followed by assembly toward the C-terminal membrane anchors, thus initiating membrane fusion. After fusion, the SNARE complex is disassembled by the AAA-ATPase N-ethylmaleimide-sensitive factor that requires the cofactor α-SNAP to first bind to the assembled SNARE complex. Using chromaffin granules and liposomes we now show that α-SNAP on its own interferes with the zippering of membrane-anchored SNARE complexes midway through the zippering reaction, arresting SNAREs in a partially assembled trans-complex and preventing fusion. Intriguingly, the interference does not result in an inhibitory effect on synaptic vesicles, suggesting that membrane properties also influence the final outcome of α-SNAP interference with SNARE zippering. We suggest that binding of α-SNAP to the SNARE complex affects the ability of the SNARE complex to harness energy or transmit force to the membrane. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Autoradiographic visualization of the mouse egg's sperm receptor bound to sperm

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bleil, J.D.; Wassarman, P.M.

1986-04-01

The extracellular coat, or zona pellucida, of mammalian eggs contains species-specific receptors to which sperm bind as a prelude to fertilization. In mice, ZP3, one of only three zona pellucida glycoproteins, serves as sperm receptor. Acrosome-intact, but not acrosome-reacted, mouse sperm recognize and interact with specific O-linked oligosaccharides of ZP3 resulting in sperm-egg binding. Binding, in turn, causes sperm to undergo the acrosome reaction; a membrane fusion event that results in loss of plasma membrane at the anterior region of the head and exposure of inner acrosomal membrane with its associated acrosomal contents. Bound, acrosome-reacted sperm are able to penetratemore » the zona pellucida and fuse with the egg's plasma membrane (fertilization). In the present report, we examined binding of radioiodinated, purified, egg ZP3 to both acrosome intact and acrosome reacted sperm by whole-mount autoradiography. Silver grains due to bound 125I-ZP3 were found localized to the acrosomal cap region of heads of acrosome-reacted sperm. Under the same conditions, 125I-fetuin bound at only background levels to heads of both acrosome-intact and -reacted sperm, and 125I-ZP2, another zona pellucida glycoprotein, bound preferentially to acrosome-reacted sperm. These results provide visual evidence that ZP3 binds preferentially and specifically to heads of acrosome intact sperm; properties expected of the mouse egg's sperm receptor.« less

Computing Curvature Sensitivity of Biomolecules in Membranes by Simulated Buckling.

PubMed

Elías-Wolff, Federico; Lindén, Martin; Lyubartsev, Alexander P; Brandt, Erik G

2018-03-13

Membrane curvature sensing, where the binding free energies of membrane-associated molecules depend on the local membrane curvature, is a key factor to modulate and maintain the shape and organization of cell membranes. However, the microscopic mechanisms are not well understood, partly due to absence of efficient simulation methods. Here, we describe a method to compute the curvature dependence of the binding free energy of a membrane-associated probe molecule that interacts with a buckled membrane, which has been created by lateral compression of a flat bilayer patch. This buckling approach samples a wide range of curvatures in a single simulation, and anisotropic effects can be extracted from the orientation statistics. We develop an efficient and robust algorithm to extract the motion of the probe along the buckled membrane surface, and evaluate its numerical properties by extensive sampling of three coarse-grained model systems: local lipid density in a curved environment for single-component bilayers, curvature preferences of individual lipids in two-component membranes, and curvature sensing by a homotrimeric transmembrane protein. The method can be used to complement experimental data from curvature partition assays and provides additional insight into mesoscopic theories and molecular mechanisms for curvature sensing.

How actin binds and assembles onto plasma membranes from Dictyostelium discoideum

PubMed Central

1988-01-01

We have shown previously (Schwartz, M. A., and E. J. Luna. 1986. J. Cell Biol. 102: 2067-2075) that actin binds with positive cooperativity to plasma membranes from Dictyostelium discoideum. Actin is polymerized at the membrane surface even at concentrations well below the critical concentration for polymerization in solution. Low salt buffer that blocks actin polymerization in solution also prevents actin binding to membranes. To further explore the relationship between actin polymerization and binding to membranes, we prepared four chemically modified actins that appear to be incapable of polymerizing in solution. Three of these derivatives also lost their ability to bind to membranes. The fourth derivative (EF actin), in which histidine-40 is labeled with ethoxyformic anhydride, binds to membranes with reduced affinity. Binding curves exhibit positive cooperativity, and cross- linking experiments show that membrane-bound actin is multimeric. Thus, binding and polymerization are tightly coupled, and the ability of these membranes to polymerize actin is dramatically demonstrated. EF actin coassembles weakly with untreated actin in solution, but coassembles well on membranes. Binding by untreated actin and EF actin are mutually competitive, indicating that they bind to the same membrane sites. Hill plots indicate that an actin trimer is the minimum assembly state required for tight binding to membranes. The best explanation for our data is a model in which actin oligomers assemble by binding to clustered membrane sites with successive monomers on one side of the actin filament bound to the membrane. Individual binding affinities are expected to be low, but the overall actin-membrane avidity is high, due to multivalency. Our results imply that extracellular factors that cluster membrane proteins may create sites for the formation of actin nuclei and thus trigger actin polymerization in the cell. PMID:3392099

De novo design of a transmembrane Zn²⁺-transporting four-helix bundle.

PubMed

Joh, Nathan H; Wang, Tuo; Bhate, Manasi P; Acharya, Rudresh; Wu, Yibing; Grabe, Michael; Hong, Mei; Grigoryan, Gevorg; DeGrado, William F

2014-12-19

The design of functional membrane proteins from first principles represents a grand challenge in chemistry and structural biology. Here, we report the design of a membrane-spanning, four-helical bundle that transports first-row transition metal ions Zn(2+) and Co(2+), but not Ca(2+), across membranes. The conduction path was designed to contain two di-metal binding sites that bind with negative cooperativity. X-ray crystallography and solid-state and solution nuclear magnetic resonance indicate that the overall helical bundle is formed from two tightly interacting pairs of helices, which form individual domains that interact weakly along a more dynamic interface. Vesicle flux experiments show that as Zn(2+) ions diffuse down their concentration gradients, protons are antiported. These experiments illustrate the feasibility of designing membrane proteins with predefined structural and dynamic properties. Copyright © 2014, American Association for the Advancement of Science.

Supported Lipid Bilayers with Phosphatidylethanolamine as the Major Component.

PubMed

Sendecki, Anne M; Poyton, Matthew F; Baxter, Alexis J; Yang, Tinglu; Cremer, Paul S

2017-11-21

Phosphatidylethanolamine (PE) is notoriously difficult to incorporate into model membrane systems, such as fluid supported lipid bilayers (SLBs), at high concentrations because of its intrinsic negative curvature. Using fluorescence-based techniques, we demonstrate that having fewer sites of unsaturation in the lipid tails leads to high-quality SLBs because these lipids help to minimize the curvature. Moreover, shorter saturated chains can help maintain the membranes in the fluid phase. Using these two guidelines, we find that up to 70 mol % PE can be incorporated into SLBs at room temperature and up to 90 mol % PE can be incorporated at 37 °C. Curiously, conditions under which three-dimensional tubules project outward from the planar surface as well as conditions under which domain formation occurs can be found. We have employed these model membrane systems to explore the ability of Ni 2+ to bind to PE. It was found that this transition metal ion binds 1000-fold tighter to PE than to phosphatidylcholine lipids. In the future, this platform could be exploited to monitor the binding of other transition metal ions or the binding of antimicrobial peptides. It could also be employed to explore the physical properties of PE-containing membranes, such as phase domain behavior and intermolecular hydrogen bonding.

Food proteins and maturation of small intestinal microvillus membranes (MVM). III. Food protein binding and MVM proteins in rats from newborn to young adult age.

PubMed

Stern, M; Gellermann, B; Wieser, H

1990-10-01

To investigate postnatal maturational profiles of functional and biochemical properties of rat small intestinal microvillus membranes (MVM), we did a longitudinal study in rats from birth to the age of 12 weeks. In parallel, we studied binding of cow's milk proteins and of the wheat gliadin peptide B 3142, as well as MVM proteins (SDS-PAGE). Changes in MVM fluidity and lipid composition exhibited early (0-4 weeks) and intermediate and late (6-12 weeks) patterns, as has been published earlier. Postnatal changes of food protein and peptide binding occurred early during the observation period, not related to weaning. There was not much further change in binding after 6-8 weeks. Developmental profiles of MVM protein and some lipid changes resembled, but did not equal, changes in food protein binding. We conclude that changes in MVM biochemical composition affect MVM binding characteristics. In particular, high molecular weight MVM proteins (susceptible to trypsin treatment) appear to play a role in postnatal maturational differences in MVM food protein binding.

High-affinity cannabinoid binding site in brain: A possible marijuana receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nye, J.S.

The mechanism by which delta{sup 9} tetrahydrocannabinol (delta{sup 9}THC), the major psychoactive component of marijuana or hashish, produces its potent psychological and physiological effects is unknown. To find receptor binding sites for THC, we designed a water-soluble analog for use as a radioligand. 5{prime}-Trimethylammonium-delta{sup 8}THC (TMA) is a positively charged analog of delta-{sup 8}THC modified on the 5{prime} carbon, a portion of the molecule not important for its psychoactivity. We have studied the binding of ({sup 3}H)-5{prime}-trimethylammonium-delta-{sup 8}THC (({sup 3}H)TMA) to rat neuronal membranes. ({sup 3}H)TMA binds saturably and reversibly to brain membranes with high affinity to apparently one classmore » of sites. Highest binding site density occurs in brain, but several peripheral organs also display specific binding. Detergent solubilizes the sites without affecting their pharmacologial properties. Molecular sieve chromatography reveals a bimodal peak of ({sup 3}H)TMA binding activity of approximately 60,000 daltons apparent molecular weight.« less

Monoclonal immunotoxin acting on the Na/sup +/ channel, with properties similar to those of a scorpion toxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barhanin, J.; Meiri, H.; Romey, G.

1985-03-01

The authors describe the properties of a monoclonal antibody against the Na/sup +/ channel. The antibody, 72.38, competitively inhibited the binding of an /sup 125/I-labeled toxin from the Brazilian scorpion Tityus serrulatus (/sup 125/I-TiTX..gamma..) to Na/sup +/ channels of rat brain membranes. The inhibition of /sup 125/I-TiTX..gamma.. binding also was observed with the solubilized Na/sup +/ channel from rat brain membranes. Antibody 72.38 antagonized /sup 125/I-TiTX..gamma.. binding to Na/sup +/ channels from different animal species (fish, avian, and mammalian) and from different tissues (electroplax, brain, heart, and muscle). Moreover, 72.38 has been used for immunofluorescence labeling of Na/sup +/ channelsmore » in rat sciatic nodes of Ranvier and cultured dorsal root ganglion cells. Electrophysiological experiments on rat muscle cells fully confirmed the similarity between TiTX..gamma.. and 72.38 seen in binding experiments. TiTX..gamma.. and 72.38 are without effect on the ion selectivity of the Na/sup +/ channel, but they both drastically change the voltage-dependence of activation and inactivation of the Na/sup +/ channel.« less

Dynamic interactions between a membrane binding protein and lipids induce fluctuating diffusivity

PubMed Central

Yamamoto, Eiji; Akimoto, Takuma; Kalli, Antreas C.; Yasuoka, Kenji; Sansom, Mark S. P.

2017-01-01

Pleckstrin homology (PH) domains are membrane-binding lipid recognition proteins that interact with phosphatidylinositol phosphate (PIP) molecules in eukaryotic cell membranes. Diffusion of PH domains plays a critical role in biological reactions on membrane surfaces. Although diffusivity can be estimated by long-time measurements, it lacks information on the short-time diffusive nature. We reveal two diffusive properties of a PH domain bound to the surface of a PIP-containing membrane using molecular dynamics simulations. One is fractional Brownian motion, attributed to the motion of the lipids with which the PH domain interacts. The other is temporally fluctuating diffusivity; that is, the short-time diffusivity of the bound protein changes substantially with time. Moreover, the diffusivity for short-time measurements is intrinsically different from that for long-time measurements. This fluctuating diffusivity results from dynamic changes in interactions between the PH domain and PIP molecules. Our results provide evidence that the complexity of protein-lipid interactions plays a crucial role in the diffusion of proteins on biological membrane surfaces. Changes in the diffusivity of PH domains and related membrane-bound proteins may in turn contribute to the formation/dissolution of protein complexes in membranes. PMID:28116358

Lipid - Motor Interactions: Soap Opera or Symphony?

PubMed

Pathak, Divya; Mallik, Roop

2017-02-01

Intracellular transport of organelles can be driven by multiple motor proteins that bind to the lipid membrane of the organelle and work as a team. We review present knowledge on how lipids orchestrate the recruitment of motors to a membrane. Looking beyond recruitment, we also discuss how heterogeneity and local mechanical properties of the membrane may influence function of motor-teams. These issues gain importance because phagocytosed pathogens use lipid-centric strategies to manipulate motors and survive in host cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

Free Energy Wells and Barriers to Ion Transport Across Membranes

NASA Astrophysics Data System (ADS)

Rempe, Susan

2014-03-01

The flow of ions across cellular membranes is essential to many biological processes. Ion transport is also important in synthetic materials used as battery electrolytes. Transport often involves specific ions and fast conduction. To achieve those properties, ion conduction pathways must solvate specific ions by just the ``right amount.'' The right amount of solvation avoids ion traps due to deep free energy wells, and avoids ion block due to high free energy barriers. Ion channel proteins in cellular membranes demonstrate this subtle balance in solvation of specific ions. Using ab initio molecular simulations, we have interrogated the link between binding site structure and ion solvation free energies in biological ion binding sites. Our results emphasize the surprisingly important role of the environment that surrounds ion-binding sites for fast transport of specific ions. We acknowledge support from Sandia's LDRD program. Sandia National Labs is a multi-program laboratory operated by Sandia Corp., a wholly owned subsidiary of Lockheed Martin Corp., for the US DOE's NNSA under contract DE-AC04-94AL85000.

Unveiling TRPV1 Spatio-Temporal Organization in Live Cell Membranes

PubMed Central

Storti, Barbara; Di Rienzo, Carmine; Cardarelli, Francesco; Bizzarri, Ranieri; Beltram, Fabio

2015-01-01

Transient Receptor Potential Vanilloid 1 (TRPV1) is a non-selective cation channel that integrates several stimuli into nociception and neurogenic inflammation. Here we investigated the subtle TRPV1 interplay with candidate membrane partners in live cells by a combination of spatio-temporal fluctuation techniques and fluorescence resonance energy transfer (FRET) imaging. We show that TRPV1 is split into three populations with fairly different molecular properties: one binding to caveolin-1 and confined into caveolar structures, one actively guided by microtubules through selective binding, and one which diffuses freely and is not directly implicated in regulating receptor functionality. The emergence of caveolin-1 as a new interactor of TRPV1 evokes caveolar endocytosis as the main desensitization pathway of TRPV1 receptor, while microtubule binding agrees with previous data suggesting the receptor stabilization in functional form by these cytoskeletal components. Our results shed light on the hitherto unknown relationships between spatial organization and TRPV1 function in live-cell membranes. PMID:25764349

Nanoparticles engineered to bind cellular motors for efficient delivery.

PubMed

Dalmau-Mena, Inmaculada; Del Pino, Pablo; Pelaz, Beatriz; Cuesta-Geijo, Miguel Ángel; Galindo, Inmaculada; Moros, María; de la Fuente, Jesús M; Alonso, Covadonga

2018-03-30

Dynein is a cytoskeletal molecular motor protein that transports cellular cargoes along microtubules. Biomimetic synthetic peptides designed to bind dynein have been shown to acquire dynamic properties such as cell accumulation and active intra- and inter-cellular motion through cell-to-cell contacts and projections to distant cells. On the basis of these properties dynein-binding peptides could be used to functionalize nanoparticles for drug delivery applications. Here, we show that gold nanoparticles modified with dynein-binding delivery sequences become mobile, powered by molecular motor proteins. Modified nanoparticles showed dynamic properties, such as travelling the cytosol, crossing intracellular barriers and shuttling the nuclear membrane. Furthermore, nanoparticles were transported from one cell to another through cell-to-cell contacts and quickly spread to distant cells through cell projections. The capacity of these motor-bound nanoparticles to spread to many cells and increasing cellular retention, thus avoiding losses and allowing lower dosage, could make them candidate carriers for drug delivery.

Theoretical Studies for Dendrimer-Based Drug Delivery.

PubMed

Bello, Martiniano; Fragoso-Vázquez, Jonathan; Correa-Basurto, José

2017-01-01

Numerous theoretical studies have been performed to iteratively optimize the physicochemical properties such as dendrimer size and surface constituents in solution, as well as their molecular recognition properties for drugs, lipid membranes, nucleic acids and proteins, etc. Molecular modeling approaches such as docking and molecular dynamic (MD) simulations have supported experimental efforts by providing important insights into the structural properties of dendrimers in solution and possible binding properties of drugs at the atomic level. We review the utilization of molecular modelling tools to obtain insight into the study and design of dendrimers, with particular importance placed on the improvement of binding properties of dendrimers for their use as drug nanocarriers and to increase the water solubility properties and drug delivery. The modeling studies discussed in this review have provided substantial insight into the physicochemical properties of dendrimers in solution, including solvent pH and counterion distribution, at the atomic level, as well as the elucidation of some of the key interactions in solution of unmodified and modified dendrimers with some drugs of pharmaceutics interest and biological systems such as nucleic acids, proteins and lipid membranes. the described studies illustrate that whether simulations will be run at the all-atom or coarse-grained level, physicochemical conditions such as the type of force field, the treatment of electrostatics effects, counterion distribution, protonation state of dendrimers, and dendrimer concentrations which have been probed to play a crucial role in the structural behavior and binding properties must be prudently incorporated in the simulations. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Monitoring glycolipid transfer protein activity and membrane interaction with the surface plasmon resonance technique.

PubMed

Ohvo-Rekilä, Henna; Mattjus, Peter

2011-01-01

The glycolipid transfer protein (GLTP) is a protein capable of binding and transferring glycolipids. GLTP is cytosolic and it can interact through its FFAT-like (two phenylalanines in an acidic tract) motif with proteins localized on the surface of the endoplasmic reticulum. Previous in vitro work with GLTP has focused mainly on the complete transfer reaction of the protein, that is, binding and subsequent removal of the glycolipid from the donor membrane, transfer through the aqueous environment, and the final release of the glycolipid to an acceptor membrane. Using bilayer vesicles and surface plasmon resonance spectroscopy, we have now, for the first time, analyzed the binding and lipid removal capacity of GLTP with a completely label-free technique. This technique is focused on the initial steps in GLTP-mediated transfer and the parameters affecting these steps can be more precisely determined. We used the new approach for detailed structure-function studies of GLTP by examining the glycolipid transfer capacity of specific GLTP tryptophan mutants. Tryptophan 96 is crucial for the transfer activity of the protein and tryptophan 142 is an important part of the proteins membrane interacting domain. Further, we varied the composition of the used lipid vesicles and gained information on the effect of membrane properties on GLTP activity. GLTP prefers to interact with more tightly packed membranes, although GLTP-mediated transfer is faster from more fluid membranes. This technique is very useful for the study of membrane-protein interactions and lipid-transfer rates and it can easily be adapted to other membrane-interacting proteins. Copyright © 2010 Elsevier B.V. All rights reserved.

Cholesterol Promotes Protein Binding by Affecting Membrane Electrostatics and Solvation Properties

DOE Office of Scientific and Technical Information (OSTI.GOV)

Doktorova, Milka; Heberle, Frederick A.; Kingston, Richard L.

Binding of the retroviral structural protein Gag to the cellular plasma membrane is mediated by the protein’s matrix (MA) domain. Prominent among MA-PM interactions is electrostatic attraction between the positively charged MA domain and the negatively charged plasma membrane inner leaflet. Previously, we reported that membrane association of HIV-1 Gag, as well as purified Rous sarcoma virus (RSV) MA and Gag, depends strongly on the presence of acidic lipids and is enhanced by cholesterol (Chol). The mechanism underlying this enhancement was unclear. Here in this paper, using a broad set of in vitro and in silico techniques we addressed molecularmore » mechanisms of association between RSV MA and model membranes, and investigated how Chol enhances this association. In neutron scattering experiments with liposomes in the presence or absence of Chol, MA preferentially interacted with preexisting POPS-rich clusters formed by nonideal lipid mixing, binding peripherally to the lipid headgroups with minimal perturbation to the bilayer structure. Molecular dynamics simulations showed a stronger MA-bilayer interaction in the presence of Chol, and a large Chol-driven increase in lipid packing and membrane surface charge density. Although in vitro MA-liposome association is influenced by disparate variables, including ionic strength and concentrations of Chol and charged lipids, continuum electrostatic theory revealed an underlying dependence on membrane surface potential. Together, these results conclusively show that Chol affects RSV MA-membrane association by making the electrostatic potential at the membrane surface more negative, while decreasing the penalty for lipid headgroup desolvation. The presented approach can be applied to other viral and nonviral proteins.« less

Cholesterol Promotes Protein Binding by Affecting Membrane Electrostatics and Solvation Properties

DOE PAGES

Doktorova, Milka; Heberle, Frederick A.; Kingston, Richard L.; ...

2017-11-07

Binding of the retroviral structural protein Gag to the cellular plasma membrane is mediated by the protein’s matrix (MA) domain. Prominent among MA-PM interactions is electrostatic attraction between the positively charged MA domain and the negatively charged plasma membrane inner leaflet. Previously, we reported that membrane association of HIV-1 Gag, as well as purified Rous sarcoma virus (RSV) MA and Gag, depends strongly on the presence of acidic lipids and is enhanced by cholesterol (Chol). The mechanism underlying this enhancement was unclear. Here in this paper, using a broad set of in vitro and in silico techniques we addressed molecularmore » mechanisms of association between RSV MA and model membranes, and investigated how Chol enhances this association. In neutron scattering experiments with liposomes in the presence or absence of Chol, MA preferentially interacted with preexisting POPS-rich clusters formed by nonideal lipid mixing, binding peripherally to the lipid headgroups with minimal perturbation to the bilayer structure. Molecular dynamics simulations showed a stronger MA-bilayer interaction in the presence of Chol, and a large Chol-driven increase in lipid packing and membrane surface charge density. Although in vitro MA-liposome association is influenced by disparate variables, including ionic strength and concentrations of Chol and charged lipids, continuum electrostatic theory revealed an underlying dependence on membrane surface potential. Together, these results conclusively show that Chol affects RSV MA-membrane association by making the electrostatic potential at the membrane surface more negative, while decreasing the penalty for lipid headgroup desolvation. The presented approach can be applied to other viral and nonviral proteins.« less

Binding constants of membrane-anchored receptors and ligands depend strongly on the nanoscale roughness of membranes.

PubMed

Hu, Jinglei; Lipowsky, Reinhard; Weikl, Thomas R

2013-09-17

Cell adhesion and the adhesion of vesicles to the membranes of cells or organelles are pivotal for immune responses, tissue formation, and cell signaling. The adhesion processes depend sensitively on the binding constant of the membrane-anchored receptor and ligand proteins that mediate adhesion, but this constant is difficult to measure in experiments. We have investigated the binding of membrane-anchored receptor and ligand proteins with molecular dynamics simulations. We find that the binding constant of the anchored proteins strongly decreases with the membrane roughness caused by thermally excited membrane shape fluctuations on nanoscales. We present a theory that explains the roughness dependence of the binding constant for the anchored proteins from membrane confinement and that relates this constant to the binding constant of soluble proteins without membrane anchors. Because the binding constant of soluble proteins is readily accessible in experiments, our results provide a useful route to compute the binding constant of membrane-anchored receptor and ligand proteins.

Effects of a detergent micelle environment on P-glycoprotein (ABCB1)-ligand interactions

PubMed Central

Shukla, Suneet; Abel, Biebele; Chufan, Eduardo E.; Ambudkar, Suresh V.

2017-01-01

P-glycoprotein (P-gp) is a multidrug transporter that uses energy from ATP hydrolysis to export many structurally dissimilar hydrophobic and amphipathic compounds, including anticancer drugs from cells. Several structural studies on purified P-gp have been reported, but only limited and sometimes conflicting information is available on ligand interactions with the isolated transporter in a dodecyl-maltoside detergent environment. In this report we compared the biochemical properties of P-gp in native membranes, detergent micelles, and when reconstituted in artificial membranes. We found that the modulators zosuquidar, tariquidar, and elacridar stimulated the ATPase activity of purified human or mouse P-gp in a detergent micelle environment. In contrast, these drugs inhibited ATPase activity in native membranes or in proteoliposomes, with IC50 values in the 10–40 nm range. Similarly, a 30–150-fold decrease in the apparent affinity for verapamil and cyclic peptide inhibitor QZ59-SSS was observed in detergent micelles compared with native or artificial membranes. Together, these findings demonstrate that the high-affinity site is inaccessible because of either a conformational change or binding of detergent at the binding site in a detergent micelle environment. The ligands bind to a low-affinity site, resulting in altered modulation of P-gp ATPase activity. We, therefore, recommend studying structural and functional aspects of ligand interactions with purified P-gp and other ATP-binding cassette transporters that transport amphipathic or hydrophobic substrates in a detergent-free native or artificial membrane environment. PMID:28283574

Stoichiometry of maltodextrin-binding sites in LamB, an outer membrane protein from Escherichia coli.

PubMed Central

Gehring, K; Cheng, C H; Nikaido, H; Jap, B K

1991-01-01

We have directly measured the stoichiometry of maltodextrin-binding sites in LamB. Scatchard plots and computer fitting of flow dialysis (rate-of-dialysis) experiments clearly establish three independent binding sites per LamB trimer, with a dissociation constant of approximately 60 microM for maltoheptaose. The current model for LamB's function as a specific pore is discussed with respect to the symmetry in LamB's kinetic properties and the implications of our results. Images PMID:2001992

Surfactant-free Colloidal Particles with Specific Binding Affinity

PubMed Central

2017-01-01

Colloidal particles with specific binding affinity are essential for in vivo and in vitro biosensing, targeted drug delivery, and micrometer-scale self-assembly. Key to these techniques are surface functionalizations that provide high affinities to specific target molecules. For stabilization in physiological environments, current particle coating methods rely on adsorbed surfactants. However, spontaneous desorption of these surfactants typically has an undesirable influence on lipid membranes. To address this issue and create particles for targeting molecules in lipid membranes, we present here a surfactant-free coating method that combines high binding affinity with stability at physiological conditions. After activating charge-stabilized polystyrene microparticles with EDC/Sulfo-NHS, we first coat the particles with a specific protein and subsequently covalently attach a dense layer of poly(ethyelene) glycol. This polymer layer provides colloidal stability at physiological conditions as well as antiadhesive properties, while the protein coating provides the specific affinity to the targeted molecule. We show that NeutrAvidin-functionalized particles bind specifically to biotinylated membranes and that Concanavalin A-functionalized particles bind specifically to the glycocortex of Dictyostelium discoideum cells. The affinity of the particles changes with protein density, which can be tuned during the coating procedure. The generic and surfactant-free coating method reported here transfers the high affinity and specificity of a protein onto colloidal polystyrene microparticles. PMID:28847149

Munc13-4 reconstitutes calcium-dependent SNARE-mediated membrane fusion

PubMed Central

Boswell, Kristin L.; James, Declan J.; Esquibel, Joseph M.; Bruinsma, Stephen; Shirakawa, Ryutaro; Horiuchi, Hisanori

2012-01-01

Munc13-4 is a widely expressed member of the CAPS/Munc13 protein family proposed to function in priming secretory granules for exocytosis. Munc13-4 contains N- and C-terminal C2 domains (C2A and C2B) predicted to bind Ca2+, but Ca2+-dependent regulation of Munc13-4 activity has not been described. The C2 domains bracket a predicted SNARE-binding domain, but whether Munc13-4 interacts with SNARE proteins is unknown. We report that Munc13-4 bound Ca2+ and restored Ca2+-dependent granule exocytosis to permeable cells (platelets, mast, and neuroendocrine cells) dependent on putative Ca2+-binding residues in C2A and C2B. Munc13-4 exhibited Ca2+-stimulated SNARE interactions dependent on C2A and Ca2+-dependent membrane binding dependent on C2B. In an apparent coupling of membrane and SNARE binding, Munc13-4 stimulated SNARE-dependent liposome fusion dependent on putative Ca2+-binding residues in both C2A and C2B domains. Munc13-4 is the first priming factor shown to promote Ca2+-dependent SNARE complex formation and SNARE-mediated liposome fusion. These properties of Munc13-4 suggest its function as a Ca2+ sensor at rate-limiting priming steps in granule exocytosis. PMID:22508512

Munc13-4 reconstitutes calcium-dependent SNARE-mediated membrane fusion.

PubMed

Boswell, Kristin L; James, Declan J; Esquibel, Joseph M; Bruinsma, Stephen; Shirakawa, Ryutaro; Horiuchi, Hisanori; Martin, Thomas F J

2012-04-16

Munc13-4 is a widely expressed member of the CAPS/Munc13 protein family proposed to function in priming secretory granules for exocytosis. Munc13-4 contains N- and C-terminal C2 domains (C2A and C2B) predicted to bind Ca(2+), but Ca(2+)-dependent regulation of Munc13-4 activity has not been described. The C2 domains bracket a predicted SNARE-binding domain, but whether Munc13-4 interacts with SNARE proteins is unknown. We report that Munc13-4 bound Ca(2+) and restored Ca(2+)-dependent granule exocytosis to permeable cells (platelets, mast, and neuroendocrine cells) dependent on putative Ca(2+)-binding residues in C2A and C2B. Munc13-4 exhibited Ca(2+)-stimulated SNARE interactions dependent on C2A and Ca(2+)-dependent membrane binding dependent on C2B. In an apparent coupling of membrane and SNARE binding, Munc13-4 stimulated SNARE-dependent liposome fusion dependent on putative Ca(2+)-binding residues in both C2A and C2B domains. Munc13-4 is the first priming factor shown to promote Ca(2+)-dependent SNARE complex formation and SNARE-mediated liposome fusion. These properties of Munc13-4 suggest its function as a Ca(2+) sensor at rate-limiting priming steps in granule exocytosis.

Fluorescence study of protein-lipid complexes with a new symmetric squarylium probe.

PubMed

Ioffe, Valeriya M; Gorbenko, Galyna P; Deligeorgiev, Todor; Gadjev, Nikolai; Vasilev, Aleksey

2007-06-01

The novel symmetric squarylium derivative SQ-1 has been synthesized and tested for its sensitivity to the formation of protein-lipid complexes. SQ-1 binding to the model membranes composed of zwitterionic lipid phosphatidylcholine (PC) and its mixtures with anionic lipid cardiolipin (CL) in different molar ratios was found to be controlled mainly by hydrophobic interactions. Lysozyme (Lz) and ribonuclease A (RNase) exerted an influence on the probe association with lipid vesicles resulting presumably from the competition between SQ-1 and the proteins for bilayer free volume and modification of its properties. The magnitude of this effect was much higher for lysozyme which may stem from the amphipathy of protein alpha-helix involved in the membrane binding. Varying membrane composition provides evidence for the dye sensitivity to both hydrophobic and electrostatic protein-lipid interactions. Fluorescence anisotropy studies uncovered the restriction of SQ-1 rotational mobility in lipid environment in the presence of Lz and RNase being indicative of the incorporation of the proteins into bilayer interior. The results of binding, fluorescence quenching and kinetic experiments suggested lysozyme-induced local lipid demixing upon protein association with negatively charged membranes with threshold concentration of CL for the lipid demixing being 10 mol%.

Lipids and lipid binding proteins: a perfect match.

PubMed

Glatz, Jan F C

2015-02-01

Lipids serve a great variety of functions, ranging from structural components of biological membranes to signaling molecules affecting various cellular functions. Several of these functions are related to the unique physico-chemical properties shared by all lipid species, i.e., their hydrophobicity. The latter, however, is accompanied by a poor solubility in an aqueous environment and thus a severe limitation in the transport of lipids in aqueous compartments such as blood plasma and the cellular soluble cytoplasm. Specific proteins which can reversibly and non-covalently associate with lipids, designated as lipid binding proteins or lipid chaperones, greatly enhance the aqueous solubility of lipids and facilitate their transport between tissues and within tissue cells. Importantly, transport of lipids across biological membranes also is facilitated by specific (membrane-associated) lipid binding proteins. Together, these lipid binding proteins determine the bio-availability of their ligands, and thereby markedly influence the subsequent processing, utilization, or signaling effect of lipids. The bio-availability of specific lipid species thus is governed by the presence of specific lipid binding proteins, the affinity of these proteins for distinct lipid species, and the presence of competing ligands (including pharmaceutical compounds). Recent studies suggest that post-translational modifications of lipid binding proteins may have great impact on lipid-protein interactions. As a result, several levels of regulation exist that together determine the bio-availability of lipid species. This short review discusses the significance of lipid binding proteins and their potential application as targets for therapeutic intervention. Copyright © 2014 Elsevier Ltd. All rights reserved.

Biophysics of α-Synuclein Membrane Interactions

PubMed Central

Pfefferkorn, Candace M.; Jiang, Zhiping; Lee, Jennifer C.

2011-01-01

Membrane proteins participate in nearly all cellular processes; however, because of experimental limitations, their characterization lags far behind that of soluble proteins. Peripheral membrane proteins are particularly challenging to study because of their inherent propensity to adopt multiple and/or transient conformations in solution and upon membrane association. In this review, we summarize useful biophysical techniques for the study of peripheral membrane proteins and their application in the characterization of the membrane interactions of the natively unfolded and Parkinson’s disease (PD) related protein, α-synuclein (α-syn). We give particular focus to studies that have led to the current understanding of membrane-bound α-syn structure and the elucidation of specific membrane properties that affect α-syn-membrane binding. Finally, we discuss biophysical evidence supporting a key role for membranes and α-syn in PD pathogenesis. PMID:21819966

Self-assembly and interactions of short antimicrobial cationic lipopeptides with membrane lipids: ITC, FTIR and molecular dynamics studies.

PubMed

Sikorska, Emilia; Dawgul, Małgorzata; Greber, Katarzyna; Iłowska, Emilia; Pogorzelska, Aneta; Kamysz, Wojciech

2014-10-01

In this work, the self-organization and the behavior of the surfactant-like peptides in the presence of biological membrane models were studied. The studies were focused on synthetic palmitic acid-containing lipopeptides, C16-KK-NH2 (I), C16-KGK-NH2 (II) and C16-KKKK-NH2 (III). The self-assembly was explored by molecular dynamics simulations using a coarse-grained force field. The critical micellar concentration was estimated by the surface tension measurements. The thermodynamics of the peptides binding to the anionic and zwitterionic lipids were established using isothermal titration calorimetry (ITC). The influence of the peptides on the lipid acyl chain ordering was determined using FTIR spectroscopy. The compounds studied show surface-active properties with a distinct CMC over the millimolar range. An increase in the steric and electrostatic repulsion between polar head groups shifts the CMC toward higher values and reduces the aggregation number. An analysis of the peptide-membrane binding revealed a unique interplay between the initial electrostatic and the subsequent hydrophobic interactions enabling the lipopeptides to interact with the lipid bilayer. In the case of C16-KKKK-NH2 (III), compensation of the electrostatic and hydrophobic interactions upon binding to the anionic membrane has been suggested and consequently no overall binding effects were noticed in ITC thermograms and FTIR spectra. Copyright © 2014 Elsevier B.V. All rights reserved.

Escherichia coli mutants impaired in maltodextrin transport.

PubMed

Wandersman, C; Schwartz, M; Ferenci, T

1979-10-01

Wild-type Escherichia coli K-12 was found to grow equally well on maltose and on maltodextrins containing up to seven glucose residues. Three classes of mutants unable to grow on maltodextrins, but still able to grow on maltose, were investigated in detail. The first class, already known, was composed of phage lambda-resistant mutants, which lack the outer membrane protein coded by gene lamB. These mutants grow on maltose and maltotriose but not at all on maltotetraose and longer maltodextrins which cannot cross the outer membrane. A second class of mutants were affected in malE, the structural gene of the periplasmic maltose binding protein. The maltose binding proteins isolated from the new mutants were altered in their substrate binding properties, but not in a way that could account for the mutant phenotypes. Rather, the results of growth experiments and transport studies suggest that these malE mutants are impaired in their ability to transport maltodextrins across the outer membrane. This implies that the maltose binding protein (in wild-type strains) cooperates with the lambda receptor in permeation through the outer membrane. The last class of mutants described in this paper were affected in malG, or perhaps in an as yet undetected gene close to malG. They were defective in the transfer of maltodextrins from the periplasmic space to the cytoplasm but only slightly affected in the transport of maltose.

Hyaluronate-binding proteins of murine brain.

PubMed

Marks, M S; Chi-Rosso, G; Toole, B P

1990-01-01

The distribution of hyaluronate-binding activity was determined in the soluble and membrane fractions derived from adult mouse brain by sonication in low-ionic-strength buffer. Approximately 60% of the total activity was recovered in the soluble fraction and 33% in membrane fractions. In both cases, the hyaluronate-binding activities were found to be of high affinity (KD = 10(-9) M), specific for hyaluronate, and glycoprotein in nature. Most of the hyaluronate-binding activity from the soluble fraction chromatographed in the void volume of Sepharose CL-4B and CL-6B. Approximately 50% of this activity was highly negatively charged, eluting from diethylaminoethyl (DEAE)-cellulose in 0.5 M NaCl, and contained chondroitin sulfate chains. This latter material also reacted with antibodies raised against cartilage link protein and the core protein of cartilage proteoglycan. Thus, the binding and physical characteristics of this hyaluronate-binding activity are consistent with those of a chondroitin sulfate proteoglycan aggregate similar to that found in cartilage. A 500-fold purification of this proteoglycan-like, hyaluronate-binding material was achieved by wheat germ agglutinin affinity chromatography, molecular sieve chromatography on Sepharose CL-6B, and ion exchange chromatography on DEAE-cellulose. Another class of hyaluronate-binding material (25-50% of that recovered) eluted from DEAE with 0.24 M NaCl; this material had the properties of a complex glycoprotein, did not contain chondroitin sulfate, and did not react with the antibodies against cartilage link protein and proteoglycan. Thus, adult mouse brain contains at least three different forms of hyaluronate-binding macromolecules. Two of these have properties similar to the link protein and proteoglycan of cartilage proteoglycan aggregates; the third is distinguishable from these entities.

MSC secretes at least 3 EV types each with a unique permutation of membrane lipid, protein and RNA.

PubMed

Lai, Ruenn Chai; Tan, Soon Sim; Yeo, Ronne Wee Yeh; Choo, Andre Boon Hwa; Reiner, Agnes T; Su, Yan; Shen, Yang; Fu, Zhiyan; Alexander, Lezhava; Sze, Siu Kwan; Lim, Sai Kiang

2016-01-01

Mesenchymal stem cell (MSC), a widely used adult stem cell candidate for regenerative medicine, has been shown to exert some of its therapeutic effects through the secretion of extracellular vesicles (EVs). These homogenously sized EVs of 100-150 ηm exhibited many exosome-like biophysical and biochemical properties and carry both proteins and RNAs. Recently, exosome-associated proteins in this MSC EV preparation were found to segregate primarily to those EVs that bind cholera toxin B chain (CTB), a GM1 ganglioside-specific ligand, and pulse-chase experiments demonstrated that these EVs have endosomal origin and carried many of the exosome-associated markers. Here, we report that only a fraction of the MSC EV proteome was found in CTB-bound EVs. Using Annexin V (AV) and Shiga toxin B subunit (ST) with affinities for phosphatidylserine and globotriaosylceramide, respectively, AV- and a ST-binding EV were identified. CTB-, AV- and ST-binding EVs all carried actin. However, the AV-binding EVs carried low or undetectable levels of the exosome-associated proteins. Only the ST-binding EVs carried RNA and EDA-containing fibronectin. Proteins in AV-binding EVs were also different from those released by apoptotic MSCs. CTB- and AV-binding activities were localized to the plasma membrane and cytoplasm of MSCs, while ST-binding activity was localized to the nucleus. Together, this study demonstrates that cells secrete many types of EVs. Specifically, MSCs secrete at least 3 types. They can be differentially isolated based on their affinities for membrane lipid-binding ligands. As the subcellular sites of the binding activities of these ligands and cargo load are different for each EV type, they are likely to have a different biogenesis pathway and possibly different functions.

Cholesterol Promotes Protein Binding by Affecting Membrane Electrostatics and Solvation Properties.

PubMed

Doktorova, Milka; Heberle, Frederick A; Kingston, Richard L; Khelashvili, George; Cuendet, Michel A; Wen, Yi; Katsaras, John; Feigenson, Gerald W; Vogt, Volker M; Dick, Robert A

2017-11-07

Binding of the retroviral structural protein Gag to the cellular plasma membrane is mediated by the protein's matrix (MA) domain. Prominent among MA-PM interactions is electrostatic attraction between the positively charged MA domain and the negatively charged plasma membrane inner leaflet. Previously, we reported that membrane association of HIV-1 Gag, as well as purified Rous sarcoma virus (RSV) MA and Gag, depends strongly on the presence of acidic lipids and is enhanced by cholesterol (Chol). The mechanism underlying this enhancement was unclear. Here, using a broad set of in vitro and in silico techniques we addressed molecular mechanisms of association between RSV MA and model membranes, and investigated how Chol enhances this association. In neutron scattering experiments with liposomes in the presence or absence of Chol, MA preferentially interacted with preexisting POPS-rich clusters formed by nonideal lipid mixing, binding peripherally to the lipid headgroups with minimal perturbation to the bilayer structure. Molecular dynamics simulations showed a stronger MA-bilayer interaction in the presence of Chol, and a large Chol-driven increase in lipid packing and membrane surface charge density. Although in vitro MA-liposome association is influenced by disparate variables, including ionic strength and concentrations of Chol and charged lipids, continuum electrostatic theory revealed an underlying dependence on membrane surface potential. Together, these results conclusively show that Chol affects RSV MA-membrane association by making the electrostatic potential at the membrane surface more negative, while decreasing the penalty for lipid headgroup desolvation. The presented approach can be applied to other viral and nonviral proteins. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Optical microwell assay of membrane transport kinetics.

PubMed

Kiskin, Nikolai I; Siebrasse, Jan P; Peters, Reiner

2003-10-01

In optical single transporter recording, membranes are firmly attached to flat solid substrates containing small wells or test compartments (TC). Transport of fluorescent molecules through TC-spanning membrane patches is induced by solution change and recorded by confocal microscopy. Previously, track-etched membrane filters were used to create solid substrates containing populations of randomly distributed TCs. In this study the possibilities offered by orderly TC arrays as created by laser microdrilling were explored. A theoretical framework was developed taking the convolution of membrane transport, solution change, and diffusion into account. The optical properties of orderly TC arrays were studied and the kinetics of solution change measured. Export and import through the nuclear pore complex (NPC) was analyzed in isolated envelopes of Xenopus oocyte nuclei. In accordance with previous reports nuclear transport receptor NTF2, which binds directly to NPC proteins, was found to be translocated much faster than "inert" molecules of similar size. Unexpectedly, NXT1, a homolog of NTF2 reportedly unable to bind to NPC proteins directly, was translocated as fast as NTF2. Thus, microstructured TC arrays were shown to provide optical single transporter recording with a new basis.

Structural basis for membrane targeting by the MVB12-associated [beta]-prism domain of the human ESCRT-I MVB12 subunit

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boura, Evzen; Hurley, James H.

2012-03-15

MVB12-associated {beta}-prism (MABP) domains are predicted to occur in a diverse set of membrane-associated bacterial and eukaryotic proteins, but their existence, structure, and biochemical properties have not been characterized experimentally. Here, we find that the MABP domains of the MVB12A and B subunits of ESCRT-I are functional modules that bind in vitro to liposomes containing acidic lipids depending on negative charge density. The MABP domain is capable of autonomously localizing to subcellular puncta and to the plasma membrane. The 1.3-{angstrom} atomic resolution crystal structure of the MVB12B MABP domain reveals a {beta}-prism fold, a hydrophobic membrane-anchoring loop, and an electropositivemore » phosphoinositide-binding patch. The basic patch is open, which explains how it senses negative charge density but lacks stereoselectivity. These observations show how ESCRT-I could act as a coincidence detector for acidic phospholipids and protein ligands, enabling it to function both in protein transport at endosomes and in cytokinesis and viral budding at the plasma membrane.« less

Biophysics of α-synuclein membrane interactions.

PubMed

Pfefferkorn, Candace M; Jiang, Zhiping; Lee, Jennifer C

2012-02-01

Membrane proteins participate in nearly all cellular processes; however, because of experimental limitations, their characterization lags far behind that of soluble proteins. Peripheral membrane proteins are particularly challenging to study because of their inherent propensity to adopt multiple and/or transient conformations in solution and upon membrane association. In this review, we summarize useful biophysical techniques for the study of peripheral membrane proteins and their application in the characterization of the membrane interactions of the natively unfolded and Parkinson's disease (PD) related protein, α-synuclein (α-syn). We give particular focus to studies that have led to the current understanding of membrane-bound α-syn structure and the elucidation of specific membrane properties that affect α-syn-membrane binding. Finally, we discuss biophysical evidence supporting a key role for membranes and α-syn in PD pathogenesis. This article is part of a Special Issue entitled: Membrane protein structure and function. Copyright © 2011. Published by Elsevier B.V.

[Modification of retinal photoreceptor membranes and Ca ion binding].

PubMed

Korchagin, V P; Berman, A L; Shukoliukov, S A; Rychkova, M P; Etingof, R N

1978-10-01

Calcium binding by modified photoreceptor membranes of cattle retina has been studied. Ca2+-binding the membranes significantly changes after C-phospholipase treatment, displaying the initial growth (less than 65% of lipid phosphorus removed) with subsequent decrease (more than 65% of phosphorus removed). Liposomes of the photoreceptor membranes lipids were found to bind more calcium than do the native photoreceptor membranes. Proteolytic enzymes (papaine, pronase) splitting some rhodopsin fragments do not affect the ability of the membrane to bind Ca2+. The increase of light-induced Ca-binding is observed only after the outer segments preincubation under conditions providing for rhodopsin phosphorylation. This effect was observed also after the splitting of the rhodopsin fragment by papaine. It is concluded that calcium binding in the photoreceptor membranes is mainly due to the phosphate groups of phospholipids.

Red blood cells serve as intravascular carriers of myeloperoxidase.

PubMed

Adam, Matti; Gajdova, Silvie; Kolarova, Hana; Kubala, Lukas; Lau, Denise; Geisler, Anne; Ravekes, Thorben; Rudolph, Volker; Tsao, Philip S; Blankenberg, Stefan; Baldus, Stephan; Klinke, Anna

2014-09-01

Myeloperoxidase (MPO) is a heme enzyme abundantly expressed in polymorphonuclear neutrophils. MPO is enzymatically capable of catalyzing the generation of reactive oxygen species (ROS) and the consumption of nitric oxide (NO). Thus MPO has both potent microbicidal and, upon binding to the vessel wall, pro-inflammatory properties. Interestingly, MPO - a highly cationic protein - has been shown to bind to both endothelial cells and leukocyte membranes. Given the anionic surface charge of red blood cells, we investigated binding of MPO to erythrocytes. Red blood cells (RBCs) derived from patients with elevated MPO plasma levels showed significantly higher amounts of MPO by flow cytometry and ELISA than healthy controls. Heparin-induced MPO-release from patient-derived RBCs was significantly increased compared to controls. Ex vivo experiments revealed dose and time dependency for MPO-RBC binding, and immunofluorescence staining as well as confocal microscopy localized MPO-RBC interaction to the erythrocyte plasma membrane. NO-consumption by RBC-membrane fragments (erythrocyte "ghosts") increased with incrementally greater concentrations of MPO during incubation, indicating preserved catalytic MPO activity. In vivo infusion of MPO-loaded RBCs into C57BL/6J mice increased local MPO tissue concentrations in liver, spleen, lung, and heart tissue as well as within the cardiac vasculature. Further, NO-dependent relaxation of aortic rings was altered by RBC bound-MPO and systemic vascular resistance significantly increased after infusion of MPO-loaded RBCs into mice. In summary, we find that MPO binds to RBC membranes in vitro and in vivo, is transported by RBCs to remote sites in mice, and affects endothelial function as well as systemic vascular resistance. RBCs may avidly bind circulating MPO, and act as carriers of this leukocyte-derived enzyme. Copyright © 2014 Elsevier Ltd. All rights reserved.

Cholesterol is necessary both for the toxic effect of Abeta peptides on vascular smooth muscle cells and for Abeta binding to vascular smooth muscle cell membranes.

PubMed

Subasinghe, Supundi; Unabia, Sharon; Barrow, Colin J; Mok, Su San; Aguilar, Marie-Isabel; Small, David H

2003-02-01

Accumulation of beta amyloid (Abeta) in the brain is central to the pathogenesis of Alzheimer's disease. Abeta can bind to membrane lipids and this binding may have detrimental effects on cell function. In this study, surface plasmon resonance technology was used to study Abeta binding to membranes. Abeta peptides bound to synthetic lipid mixtures and to an intact plasma membrane preparation isolated from vascular smooth muscle cells. Abeta peptides were also toxic to vascular smooth muscle cells. There was a good correlation between the toxic effect of Abeta peptides and their membrane binding. 'Ageing' the Abeta peptides by incubation for 5 days increased the proportion of oligomeric species, and also increased toxicity and the amount of binding to lipids. The toxicities of various Abeta analogs correlated with their lipid binding. Significantly, binding was influenced by the concentration of cholesterol in the lipid mixture. Reduction of cholesterol in vascular smooth muscle cells not only reduced the binding of Abeta to purified plasma membrane preparations but also reduced Abeta toxicity. The results support the view that Abeta toxicity is a direct consequence of binding to lipids in the membrane. Reduction of membrane cholesterol using cholesterol-lowering drugs may be of therapeutic benefit because it reduces Abeta-membrane binding.

Surface plasmon resonance spectroscopy studies of membrane proteins: transducin binding and activation by rhodopsin monitored in thin membrane films.

PubMed Central

Salamon, Z; Wang, Y; Soulages, J L; Brown, M F; Tollin, G

1996-01-01

Surface plasmon resonance (SPR) spectroscopy can provide useful information regarding average structural properties of membrane films supported on planar solid substrates. Here we have used SPR spectroscopy for the first time to monitor the binding and activation of G-protein (transducin or Gt) by bovine rhodopsin incorporated into an egg phosphatidylcholine bilayer deposited on a silver film. Rhodopsin incorporation into the membrane, performed by dilution of a detergent solution of the protein, proceeds in a saturable manner. Before photolysis, the SPR data show that Gt binds tightly (Keq approximately equal to 60 nM) and with positive cooperativity to rhodopsin in the lipid layer to form a closely packed film. A simple multilayer model yields a calculated average thickness of about 57 A, in good agreement with the structure of Gt. The data also demonstrate that Gt binding saturates at a Gt/rhodopsin ratio of approximately 0.6. Moreover, upon visible light irradiation, characteristic changes occur in the SPR spectrum, which can be modeled by a 6 A increase in the average thickness of the lipid/protein film caused by formation of metarhodopsin II (MII). Upon subsequent addition of GTP, further SPR spectral changes are induced. These are interpreted as resulting from dissociation of the alpha-subunit of Gt, formation of new MII-Gt complexes, and possible conformational changes of Gt as a consequence of complex formation. The above results clearly demonstrate the ability of SPR spectroscopy to monitor interactions among the proteins associated with signal transduction in membrane-bound systems. Images FIGURE 1 PMID:8804611

The cholesterol-dependent cytolysins pneumolysin and streptolysin O require binding to red blood cell glycans for hemolytic activity

PubMed Central

Shewell, Lucy K.; Harvey, Richard M.; Higgins, Melanie A.; Day, Christopher J.; Hartley-Tassell, Lauren E.; Chen, Austen Y.; Gillen, Christine M.; James, David B. A.; Alonzo, Francis; Torres, Victor J.; Walker, Mark J.; Paton, Adrienne W.; Paton, James C.; Jennings, Michael P.

2014-01-01

The cholesterol-dependent cytolysin (CDC) pneumolysin (Ply) is a key virulence factor of Streptococcus pneumoniae. Membrane cholesterol is required for the cytolytic activity of this toxin, but it is not clear whether cholesterol is the only cellular receptor. Analysis of Ply binding to a glycan microarray revealed that Ply has lectin activity and binds glycans, including the Lewis histo-blood group antigens. Surface plasmon resonance analysis showed that Ply has the highest affinity for the sialyl LewisX (sLeX) structure, with a Kd of 1.88 × 10−5 M. Ply hemolytic activity against human RBCs showed dose-dependent inhibition by sLeX. Flow cytometric analysis and Western blots showed that blocking binding of Ply to the sLeX glycolipid on RBCs prevents deposition of the toxin in the membrane. The lectin domain responsible for sLeX binding is in domain 4 of Ply, which contains candidate carbohydrate-binding sites. Mutagenesis of these predicted carbohydrate-binding residues of Ply resulted in a decrease in hemolytic activity and a reduced affinity for sLeX. This study reveals that this archetypal CDC requires interaction with the sLeX glycolipid cellular receptor as an essential step before membrane insertion. A similar analysis conducted on streptolysin O from Streptococcus pyogenes revealed that this CDC also has glycan-binding properties and that hemolytic activity against RBCs can be blocked with the glycan lacto-N-neotetraose by inhibiting binding to the cell surface. Together, these data support the emerging paradigm shift that pore-forming toxins, including CDCs, have cellular receptors other than cholesterol that define target cell tropism. PMID:25422425

The cholesterol-dependent cytolysins pneumolysin and streptolysin O require binding to red blood cell glycans for hemolytic activity.

PubMed

Shewell, Lucy K; Harvey, Richard M; Higgins, Melanie A; Day, Christopher J; Hartley-Tassell, Lauren E; Chen, Austen Y; Gillen, Christine M; James, David B A; Alonzo, Francis; Torres, Victor J; Walker, Mark J; Paton, Adrienne W; Paton, James C; Jennings, Michael P

2014-12-09

The cholesterol-dependent cytolysin (CDC) pneumolysin (Ply) is a key virulence factor of Streptococcus pneumoniae. Membrane cholesterol is required for the cytolytic activity of this toxin, but it is not clear whether cholesterol is the only cellular receptor. Analysis of Ply binding to a glycan microarray revealed that Ply has lectin activity and binds glycans, including the Lewis histo-blood group antigens. Surface plasmon resonance analysis showed that Ply has the highest affinity for the sialyl LewisX (sLeX) structure, with a K(d) of 1.88 × 10(-5) M. Ply hemolytic activity against human RBCs showed dose-dependent inhibition by sLeX. Flow cytometric analysis and Western blots showed that blocking binding of Ply to the sLeX glycolipid on RBCs prevents deposition of the toxin in the membrane. The lectin domain responsible for sLeX binding is in domain 4 of Ply, which contains candidate carbohydrate-binding sites. Mutagenesis of these predicted carbohydrate-binding residues of Ply resulted in a decrease in hemolytic activity and a reduced affinity for sLeX. This study reveals that this archetypal CDC requires interaction with the sLeX glycolipid cellular receptor as an essential step before membrane insertion. A similar analysis conducted on streptolysin O from Streptococcus pyogenes revealed that this CDC also has glycan-binding properties and that hemolytic activity against RBCs can be blocked with the glycan lacto-N-neotetraose by inhibiting binding to the cell surface. Together, these data support the emerging paradigm shift that pore-forming toxins, including CDCs, have cellular receptors other than cholesterol that define target cell tropism.

Interaction of SR 33557 with skeletal muscle calcium channel blocker receptors in the baboon: characterization of its binding sites

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sol-Rolland, J.; Joseph, M.; Rinaldi-Carmona, M.

1991-05-01

A procedure for the isolation of primate skeletal microsomal membranes was initiated. Membranes exhibited specific enzymatic markers such as 5'-nucleotidase, Ca{sup 2}{sup +},Mg({sup 2}{sup +})-adenosine triphosphatase and an ATP-dependent calcium uptake. Baboon skeletal microsomes bound specifically with high-affinity potent Ca{sup 2}{sup +} channel blockers such as dihydropyridine, phenylalkylamine and benzothiazepine derivatives. Scatchard analysis of equilibrium binding assays with ({sup 3}H)(+)-PN 200-110, ({sup 3}H)(-)-desmethoxyverapamil (( {sup 3}H)(-)-D888) and ({sup 3}H)-d-cis-dilitiazem were consistent with a single class of binding sites for the three radioligands. The pharmacological profile of SR 33557, an original compound with calcium antagonist properties, was investigated using radioligand bindingmore » studies. SR 33557 totally inhibited the specific binding of the three main classes of Ca{sup 2}{sup +} channel effectors and interacted allosterically with them. In addition, SR 33557 bound with high affinity to a homogeneous population of binding sites in baboon skeletal muscle.« less

Reconstitution of high affinity. cap alpha. /sub 2/ adrenergic agonist binding by fusion with a pertussis toxin substrate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, M.H.; Neubig, R.R.

1986-03-05

High affinity ..cap alpha../sub 2/ adrenergic agonist binding is thought to occur via a coupling of the ..cap alpha../sub 2/ receptor with N/sub i/, the inhibitory guanyl nucleotide binding protein. Human platelet membranes pretreated at pH 11.5 exhibit a selective inactivation of agonist binding and N/sub i/. To further study the mechanism of agonist binding, alkali treated membranes (ATM) were mixed with membranes pretreated with 10 ..mu..M phenoxybenzamine to block ..cap alpha../sub 2/ receptors (POB-M). The combined membrane pellet was incubated in 50% polyethylene glycol (PEG) to promote membrane-membrane fusion and assayed for binding to the ..cap alpha../sub 2/ agonistmore » (/sup 3/H)UK 14,304 (UK) and the antagonist (/sup 3/H) yohimbine. PEG treatment resulted in a 2-4 fold enhancement of UK binding whereas yohimbine binding was unchanged. No enhancement of UK binding was observed in the absence of PEG treatment. The reconstitution was dependent on the addition of POB-M. They found that a 1:1 ratio of POB-M:ATM was optimal. Reconstituted binding was inhibited by GppNHp. Fusion of rat C6 glioma cell membranes, which do not contain ..cap alpha../sub 2/ receptors, also enhanced agonist binding to ATM. Fusion of C6 membranes from cells treated with pertussis toxin did not enhance (/sup 3/H) UK binding. These data show that a pertussis toxin sensitive membrane component, possibly N/sub i/, can reconstitute high affinity ..cap alpha../sub 2/ agonist binding.« less

Multiple Surface Regions on the Niemann-Pick C2 Protein Facilitate Intracellular Cholesterol Transport.

PubMed

McCauliff, Leslie A; Xu, Zhi; Li, Ran; Kodukula, Sarala; Ko, Dennis C; Scott, Matthew P; Kahn, Peter C; Storch, Judith

2015-11-06

The cholesterol storage disorder Niemann-Pick type C (NPC) disease is caused by defects in either of two late endosomal/lysosomal proteins, NPC1 and NPC2. NPC2 is a 16-kDa soluble protein that binds cholesterol in a 1:1 stoichiometry and can transfer cholesterol between membranes by a mechanism that involves protein-membrane interactions. To examine the structural basis of NPC2 function in cholesterol trafficking, a series of point mutations were generated across the surface of the protein. Several NPC2 mutants exhibited deficient sterol transport properties in a set of fluorescence-based assays. Notably, these mutants were also unable to promote egress of accumulated intracellular cholesterol from npc2(-/-) fibroblasts. The mutations mapped to several regions on the protein surface, suggesting that NPC2 can bind to more than one membrane simultaneously. Indeed, we have previously demonstrated that WT NPC2 promotes vesicle-vesicle interactions. These interactions were abrogated, however, by mutations causing defective sterol transfer properties. Molecular modeling shows that NPC2 is highly plastic, with several intense positively charged regions across the surface that could interact favorably with negatively charged membrane phospholipids. The point mutations generated in this study caused changes in NPC2 surface charge distribution with minimal conformational changes. The plasticity, coupled with membrane flexibility, probably allows for multiple cholesterol transfer routes. Thus, we hypothesize that, in part, NPC2 rapidly traffics cholesterol between closely appositioned membranes within the multilamellar interior of late endosomal/lysosomal proteins, ultimately effecting cholesterol egress from this compartment. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Single DNA molecules on freestanding and supported cationic lipid bilayers: diverse conformational dynamics controlled by the local bilayer properties

NASA Astrophysics Data System (ADS)

Herold, Christoph; Schwille, Petra; Petrov, Eugene P.

2016-02-01

We present experimental results on the interaction of DNA macromolecules with cationic lipid membranes with different properties, including freestanding membranes in the fluid and gel state, and supported lipid membranes in the fluid state and under conditions of fluid-gel phase coexistence. We observe diverse conformational dynamics of membrane-bound DNA molecules controlled by the local properties of the lipid bilayer. In case of fluid-state freestanding lipid membranes, the behaviour of DNA on the membrane is controlled by the membrane charge density: whereas DNA bound to weakly charged membranes predominantly behaves as a 2D random coil, an increase in the membrane charge density leads to membrane-driven irreversible DNA collapse and formation of subresolution-sized DNA globules. On the other hand, electrostatic binding of DNA macromolecules to gel-state freestanding membranes leads to completely arrested diffusion and conformational dynamics of membrane-adsorbed DNA. A drastically different picture is observed in case of DNA interaction with supported cationic lipid bilayers: When the supported bilayer is in the fluid state, membrane-bound DNA molecules undergo 2D translational Brownian motion and conformational fluctuations, irrespectively of the charge density of the supported bilayer. At the same time, when the supported cationic membrane shows fluid-gel phase coexistence, membrane-bound DNA molecules are strongly attracted to micrometre-sized gel-phase domains enriched with the cationic lipid, which results in 2D compaction of the membrane-bound macromolecules. This DNA compaction, however, is fully reversible, and disappears as soon as the membrane is heated above the fluid-gel coexistence. We also discuss possible biological implications of our experimental findings.

Sensing Membrane Stresses by Protein Insertions

PubMed Central

Campelo, Felix; Kozlov, Michael M.

2014-01-01

Protein domains shallowly inserting into the membrane matrix are ubiquitous in peripheral membrane proteins involved in various processes of intracellular membrane shaping and remodeling. It has been suggested that these domains sense membrane curvature through their preferable binding to strongly curved membranes, the binding mechanism being mediated by lipid packing defects. Here we make an alternative statement that shallow protein insertions are universal sensors of the intra-membrane stresses existing in the region of the insertion embedding rather than sensors of the curvature per se. We substantiate this proposal computationally by considering different independent ways of the membrane stress generation among which some include changes of the membrane curvature whereas others do not alter the membrane shape. Our computations show that the membrane-binding coefficient of shallow protein insertions is determined by the resultant stress independently of the way this stress has been produced. By contrast, consideration of the correlation between the insertion binding and the membrane curvature demonstrates that the binding coefficient either increases or decreases with curvature depending on the factors leading to the curvature generation. To validate our computational model, we treat quantitatively the experimental results on membrane binding by ALPS1 and ALPS2 motifs of ArfGAP1. PMID:24722359

Characterization of the proton binding sites of extracellular polymeric substances in an anaerobic membrane bioreactor.

PubMed

Liu, Yi; Chang, Sheng; Defersha, Fantahun M

2015-07-01

This paper focuses on the characterization of the chemical compositions and acidic constants of the extracellular polymeric substances (EPSs) in an anaerobic membrane bioreactor treating synthetic brewery wastewater by using chemical analysis, linear programming analysis (LPA) of titration data, and FT-IR analysis. The linear programming analysis of titration data revealed that the EPSs have proton binding sites with pKa values from pKa ≤ 6, between 6 and 7, and approximately 9.8. The strong acidic sites (pKa ≤ 6) and some weak acidic sites (7.5 < pKa < 9.0) were found to be readily removed by 0.45-μm membrane filtration. In addition, the FT-IR analysis confirmed the presence of proteins, carbohydrates, nucleic acids, and lipids in the EPS samples. Based on the FT-IR analysis and the main chemical functional groups at the bacterial cell surfaces, the identified proton binding sites were related to carboxyl, phosphate, and hydroxyl/amine groups with pKa values of 4.6 ± 0.7, 6.6 ± 0.01, and 9.7 ± 0.1, respectively, with the corresponding respective intensities of 0.31 ± 0.05, 0.96 ± 0.3, and 1.53 ± 0.3 mmole/g-EPS. The pKa values and intensities of the proton binding sites are the fundamental molecular properties of EPSs that affect the EPS charge, molecular interactions, and metal complexation characteristics. Determination of such properties can advance Derjaguin-Landau-Verwey-Overbeek (DLVO)-based concentration polarization modeling, facilitate the estimation of the osmotic pressure of the EPS concentration polarization layers, and lead to a deeper understanding of the role of metal complexation in membrane fouling. Copyright © 2015 Elsevier Ltd. All rights reserved.

Impact of membrane curvature on amyloid aggregation.

PubMed

Terakawa, Mayu S; Lin, Yuxi; Kinoshita, Misaki; Kanemura, Shingo; Itoh, Dai; Sugiki, Toshihiko; Okumura, Masaki; Ramamoorthy, Ayyalusamy; Lee, Young-Ho

2018-04-28

The misfolding, amyloid aggregation, and fibril formation of intrinsically disordered proteins/peptides (or amyloid proteins) have been shown to cause a number of disorders. The underlying mechanisms of amyloid fibrillation and structural properties of amyloidogenic precursors, intermediates, and amyloid fibrils have been elucidated in detail; however, in-depth examinations on physiologically relevant contributing factors that induce amyloidogenesis and lead to cell death remain challenging. A large number of studies have attempted to characterize the roles of biomembranes on protein aggregation and membrane-mediated cell death by designing various membrane components, such as gangliosides, cholesterol, and other lipid compositions, and by using various membrane mimetics, including liposomes, bicelles, and different types of lipid-nanodiscs. We herein review the dynamic effects of membrane curvature on amyloid generation and the inhibition of amyloidogenic proteins and peptides, and also discuss how amyloid formation affects membrane curvature and integrity, which are key for understanding relationships with cell death. Small unilamellar vesicles with high curvature and large unilamellar vesicles with low curvature have been demonstrated to exhibit different capabilities to induce the nucleation, amyloid formation, and inhibition of amyloid-β peptides and α-synuclein. Polymorphic amyloidogenesis in small unilamellar vesicles was revealed and may be viewed as one of the generic properties of interprotein interaction-dominated amyloid formation. Several mechanical models and phase diagrams are comprehensively shown to better explain experimental findings. The negative membrane curvature-mediated mechanisms responsible for the toxicity of pancreatic β cells by the amyloid aggregation of human islet amyloid polypeptide (IAPP) and binding of the precursors of the semen-derived enhancer of viral infection (SEVI) are also described. The curvature-dependent binding modes of several types of islet amyloid polypeptides with high-resolution NMR structures are also discussed. Copyright © 2018 Elsevier B.V. All rights reserved.

Peptide:lipid ratio and membrane surface charge determine the mechanism of action of the antimicrobial peptide BP100. Conformational and functional studies.

PubMed

Manzini, Mariana C; Perez, Katia R; Riske, Karin A; Bozelli, José C; Santos, Talita L; da Silva, Marcia A; Saraiva, Greice K V; Politi, Mario J; Valente, Ana P; Almeida, Fábio C L; Chaimovich, Hernan; Rodrigues, Magali A; Bemquerer, Marcelo P; Schreier, Shirley; Cuccovia, Iolanda M

2014-07-01

The cecropin-melittin hybrid antimicrobial peptide BP100 (H-KKLFKKILKYL-NH2) is selective for Gram-negative bacteria, negatively charged membranes, and weakly hemolytic. We studied BP100 conformational and functional properties upon interaction with large unilamellar vesicles, LUVs, and giant unilamellar vesicles, GUVs, containing variable proportions of phosphatidylcholine (PC) and negatively charged phosphatidylglycerol (PG). CD and NMR spectra showed that upon binding to PG-containing LUVs BP100 acquires α-helical conformation, the helix spanning residues 3-11. Theoretical analyses indicated that the helix is amphipathic and surface-seeking. CD and dynamic light scattering data evinced peptide and/or vesicle aggregation, modulated by peptide:lipid ratio and PG content. BP100 decreased the absolute value of the zeta potential (ζ) of LUVs with low PG contents; for higher PG, binding was analyzed as an ion-exchange process. At high salt, BP100-induced LUVS leakage requires higher peptide concentration, indicating that both electrostatic and hydrophobic interactions contribute to peptide binding. While a gradual release took place at low peptide:lipid ratios, instantaneous loss occurred at high ratios, suggesting vesicle disruption. Optical microscopy of GUVs confirmed BP100-promoted disruption of negatively charged membranes. The mechanism of action of BP100 is determined by both peptide:lipid ratio and negatively charged lipid content. While gradual release results from membrane perturbation by a small number of peptide molecules giving rise to changes in acyl chain packing, lipid clustering (leading to membrane defects), and/or membrane thinning, membrane disruption results from a sequence of events - large-scale peptide and lipid clustering, giving rise to peptide-lipid patches that eventually would leave the membrane in a carpet-like mechanism. Copyright © 2014 Elsevier B.V. All rights reserved.

A Brain Membrane Protein Similar to the Rat src Gene Product

NASA Astrophysics Data System (ADS)

Scheinberg, David A.; Strand, Mette

1981-01-01

We report the purification to homogeneity of a 20,000-dalton, transformation-related, rat cell membrane protein. This protein, p20, was originally identified in preparations of a defective woolly monkey leukemia virus pseudotype of Kirsten sarcoma virus. The chromatographically purified p20 was an acidic hydrophobic protein, capable of specifically binding GTP (dissociation constant = 15 μ M). This nucleotide binding property and other previously reported characteristics were similar to properties ascribed to the Harvey sarcoma virus src gene product. p20 also appeared similar to this src gene product when immunoprecipitates of both proteins were directly compared by one- and two-dimensional NaDodSO4 gel electrophoreses. However, the proteins were not identical, because their tryptic maps differed. Using a competition radioimmunoassay, we have measured the concentration of p20 in cells, viruses, and rat tissues: p20 was not encoded by rat sarcoma viruses because it was increased only slightly after Kirsten sarcoma virus transformation of rat cells and was not increased in nonrat cells transformed by the Kirsten or Harvey sarcoma virus. Remarkably, of 10 rat tissues examined, p20 was found predominantly in brain, specifically in the membranes.

Non-Brownian diffusion in lipid membranes: Experiments and simulations.

PubMed

Metzler, R; Jeon, J-H; Cherstvy, A G

2016-10-01

The dynamics of constituents and the surface response of cellular membranes-also in connection to the binding of various particles and macromolecules to the membrane-are still a matter of controversy in the membrane biophysics community, particularly with respect to crowded membranes of living biological cells. We here put into perspective recent single particle tracking experiments in the plasma membranes of living cells and supercomputing studies of lipid bilayer model membranes with and without protein crowding. Special emphasis is put on the observation of anomalous, non-Brownian diffusion of both lipid molecules and proteins embedded in the lipid bilayer. While single component, pure lipid bilayers in simulations exhibit only transient anomalous diffusion of lipid molecules on nanosecond time scales, the persistence of anomalous diffusion becomes significantly longer ranged on the addition of disorder-through the addition of cholesterol or proteins-and on passing of the membrane lipids to the gel phase. Concurrently, experiments demonstrate the anomalous diffusion of membrane embedded proteins up to macroscopic time scales in the minute time range. Particular emphasis will be put on the physical character of the anomalous diffusion, in particular, the occurrence of ageing observed in the experiments-the effective diffusivity of the measured particles is a decreasing function of time. Moreover, we present results for the time dependent local scaling exponent of the mean squared displacement of the monitored particles. Recent results finding deviations from the commonly assumed Gaussian diffusion patterns in protein crowded membranes are reported. The properties of the displacement autocorrelation function of the lipid molecules are discussed in the light of their appropriate physical anomalous diffusion models, both for non-crowded and crowded membranes. In the last part of this review we address the upcoming field of membrane distortion by elongated membrane-binding particles. We discuss how membrane compartmentalisation and the particle-membrane binding energy may impact the dynamics and response of lipid membranes. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Characterization of Rose Bengal binding to sinusoidal and bile canalicular plasma membrane from rat liver.

PubMed

Yachi, K; Sugiyama, Y; Sawada, Y; Iga, T; Ikeda, Y; Toda, G; Hanano, M

1989-01-16

The binding of Rose bengal, a model organic anion, to sinusoidal and bile canalicular membrane fractions isolated from rat liver was compared. The fluorescence change of Rose bengal after being bound to liver plasma membranes was utilized for measuring the binding. The dissociation constants (Kd = 0.1-0.12 microM) and the binding capacities (n = 11-15 nmol/mg protein) for Rose bengal are comparable between the two membrane fractions, although the n value for sinusoidal membrane is somewhat larger than that for bile canalicular membrane. The Rose bengal binding to both membrane fractions was inhibited by various organic anions at relatively low concentrations, i.e., the half-inhibition concentrations (IC50) for Indocyanine green, sulfobromophthalein, Bromophenol blue and 1-anilino-8-naphthalene sulfonate were 0.1, 100, 1.5-2.5 and 100 microM, respectively, while taurocholate did not inhibit the Rose bengal binding to either membrane fraction at these low concentration ranges. The type of inhibition of sulfobromophthalein and Indocyanine green for Rose bengal binding is different between the two membrane domains. That is, in sinusoidal and bile canalicular membrane fractions, these organic anions exhibit mixed-type and competitive-type inhibition, respectively. It was suggested that the fluorescence method using Rose bengal may provide a simple method for detecting the specific organic anion binding protein(s) in the liver plasma membrane.

Fusion of Legionella pneumophila outer membrane vesicles with eukaryotic membrane systems is a mechanism to deliver pathogen factors to host cell membranes.

PubMed

Jäger, Jens; Keese, Susanne; Roessle, Manfred; Steinert, Michael; Schromm, Andra B

2015-05-01

The formation and release of outer membrane vesicles (OMVs) is a phenomenon observed in many bacteria, including Legionella pneumophila. During infection, this human pathogen primarily invades alveolar macrophages and replicates within a unique membrane-bound compartment termed Legionella-containing vacuole. In the current study, we analysed the membrane architecture of L. pneumophila OMVs by small-angle X-ray scattering and biophysically characterized OMV membranes. We investigated the interaction of L. pneumophila OMVs with model membranes by Förster resonance energy transfer and Fourier transform infrared spectroscopy. These experiments demonstrated the incorporation of OMV membrane material into liposomes composed of different eukaryotic phospholipids, revealing an endogenous property of OMVs to fuse with eukaryotic membranes. Cellular co-incubation experiments showed a dose- and time-dependent binding of fluorophore-labelled OMVs to macrophages. Trypan blue quenching experiments disclosed a rapid internalization of OMVs into macrophages at 37 and 4 °C. Purified OMVs induced tumour necrosis factor-α production in human macrophages at concentrations starting at 300 ng ml(-1). Experiments on HEK293-TLR2 and TLR4/MD-2 cell lines demonstrated a dominance of TLR2-dependent signalling pathways. In summary, we demonstrate binding, internalization and biological activity of L. pneumophila OMVs on human macrophages. Our data support OMV membrane fusion as a mechanism for the remote delivery of virulence factors to host cells. © 2014 John Wiley & Sons Ltd.

Calcium-Binding Properties of Sarcoplasmic Reticulum As Influenced by ATP, Caffeine, Quinine, and Local Anesthetics

PubMed Central

Carvalho, Arselio P.

1968-01-01

Calcium retained at binding sites of the sarcoplasmic reticulum membranes isolated from rabbit skeletal muscle requires 10-5 – 10-4 M ATP to exchange with 45Ca added to the medium. The ATP requirement for Ca exchangeability was observed with respect to the "intrinsic" Ca of the reticulum membranes and the fraction of Ca that is "actively" bound in the presence of ATP. Furthermore, a concentration of free Ca in the medium higher than 10-8 M is required for ATP to promote Ca exchangeability. This exchangeability is not influenced by caffeine, quinine, procaine, and tetracaine, and Ca that is either nonexchangeable (in the absence of ATP) or exchangeable (in the presence of ATP) is released by 1–5 mM quinine or tetracaine, but neither caffeine (6 mM) nor procaine (2–5 mM) has this effect. Quinine or tetracaine also releases Ca and Mg bound passively to the reticulum membranes. A possible role of ATP in maintaining the integrity of cellular membranes is discussed, and the effects of caffeine, quinine, and of local anesthetics on the binding of Ca by the isolated reticulum are related to the effects of these agents on 45Ca fluxes and on the twitch output observed in whole muscles. PMID:19873636

Redesigned Spider Peptide with Improved Antimicrobial and Anticancer Properties.

PubMed

Troeira Henriques, Sónia; Lawrence, Nicole; Chaousis, Stephanie; Ravipati, Anjaneya S; Cheneval, Olivier; Benfield, Aurélie H; Elliott, Alysha G; Kavanagh, Angela Maria; Cooper, Matthew A; Chan, Lai Yue; Huang, Yen-Hua; Craik, David J

2017-09-15

Gomesin, a disulfide-rich antimicrobial peptide produced by the Brazilian spider Acanthoscurria gomesiana, has been shown to be potent against Gram-negative bacteria and to possess selective anticancer properties against melanoma cells. In a recent study, a backbone cyclized analogue of gomesin was shown to be as active but more stable than its native form. In the current study, we were interested in improving the antimicrobial properties of the cyclic gomesin, understanding its selectivity toward melanoma cells and elucidating its antimicrobial and anticancer mode of action. Rationally designed analogues of cyclic gomesin were examined for their antimicrobial potency, selectivity toward cancer cells, membrane-binding affinity, and ability to disrupt cell and model membranes. We improved the activity of cyclic gomesin by ∼10-fold against tested Gram-negative and Gram-positive bacteria without increasing toxicity to human red blood cells. In addition, we showed that gomesin and its analogues are more toxic toward melanoma and leukemia cells than toward red blood cells and act by selectively targeting and disrupting cancer cell membranes. Preference toward some cancer types is likely dependent on their different cell membrane properties. Our findings highlight the potential of peptides as antimicrobial and anticancer leads and the importance of selectively targeting cancer cell membranes for drug development.

Backbone dynamics of the antifungal Psd1 pea defensin and its correlation with membrane interaction by NMR spectroscopy.

PubMed

de Medeiros, Luciano Neves; Angeli, Renata; Sarzedas, Carolina G; Barreto-Bergter, Eliana; Valente, Ana Paula; Kurtenbach, Eleonora; Almeida, Fabio C L

2010-02-01

Plant defensins are cysteine-rich cationic peptides, components of the innate immune system. The antifungal sensitivity of certain exemplars was correlated to the level of complex glycosphingolipids in the membrane of fungi strains. Psd1 is a 46 amino acid residue defensin isolated from pea seeds which exhibit antifungal activity. Its structure is characterized by the so-called cysteine-stabilized alpha/beta motif linked by three loops as determined by two-dimensional NMR. In the present work we explored the measurement of heteronuclear Nuclear Overhauser Effects, R1 and R2 (15)N relaxation ratios, and chemical shift to probe the backbone dynamics of Psd1 and its interaction with membrane mimetic systems with phosphatidylcholine (PC) or dodecylphosphocholine (DPC) with glucosylceramide (CMH) isolated from Fusarium solani. The calculated R2 values predicted a slow motion around the highly conserved among Gly12 residue and also in the region of the Turn3 His36-Trp38. The results showed that Psd1 interacts with vesicles of PC or PC:CMH in slightly different forms. The interaction was monitored by chemical shift perturbation and relaxation properties. Using this approach we could map the loops as the binding site of Psd1 with the membrane. The major binding epitope showed conformation exchange properties in the mus-ms timescale supporting the conformation selection as the binding mechanism. Moreover, the peptide corresponding to part of Loop1 (pepLoop1: Gly12 to Ser19) is also able to interact with DPC micelles acquiring a stable structure and in the presence of DPC:CMH the peptide changes to an extended conformation, exhibiting NOE mainly with the carbohydrate and ceramide parts of CMH. Copyright 2009 Elsevier B.V. All rights reserved.

Comparison of cannabinoid binding sites in guinea-pig forebrain and small intestine

PubMed Central

Ross, Ruth A; Brockie, Heather C; Fernando, Susanthi R; Saha, Bijali; Razdan, Raj K; Pertwee, Roger G

1998-01-01

We have investigated the nature of cannabinoid receptors in guinea-pig small intestine by establishing whether this tissue contains cannabinoid receptors with similar binding properties to those of brain CB1 receptors. The cannabinoids used were the CB1-selective antagonist SR141716A, the CB2-selective antagonist SR144528, the novel cannabinoid receptor ligand, 6′-azidohex-2′-yne-Δ8-tetrahydrocannabinol (O-1184), and the agonists CP55940, which binds equally well to CB1 and CB2 receptors, and WIN55212-2, which shows marginal CB2 selectivity.[3H]-CP55940 (1 nM) underwent extensive specific binding both to forebrain membranes (76.3%) and to membranes obtained by sucrose density gradient fractionation of homogenates of myenteric plexus-longitudinal muscle of guinea-pig small intestine (65.2%).Its binding capacity (Bmax) was higher in forebrain (4281 fmol mg−1) than in intestinal membranes (2092 fmol mg−1). However, the corresponding KD values were not significantly different from each other (2.29 and 1.75 nM respectively). Nor did the Ki values for its displacement by CP55940, WIN55212-2, O-1184, SR141716A and SR144528 from forebrain membranes (0.87, 4.15, 2.85, 5.32 and 371.9 respectively) differ significantly from the corresponding Ki values determined in experiments with intestinal membranes (0.99, 5.03, 3.16, 4.95 and 361.5 nM respectively).The Bmax values of [3H]-CP55940 and [3H]-SR141716A in forebrain membranes did not differ significantly from each other (4281 and 5658 fmol mg−1) but were both greater than the Bmax of [3H]-WIN55212-2 (2032 fmol mg−1).O-1184 (10 or 100 nM) produced parallel dextral shifts in the log concentration-response curves of WIN55212-2 and CP55940 for inhibition of electrically-evoked contractions of the myenteric plexus-longitudinal muscle preparation, its KD values being 0.20 nM (against WIN55212-2) and 0.89 nM (against CP55940).We conclude that cannabinoid binding sites in guinea-pig small intestine closely resemble CB1 binding sites of guinea-pig brain and that O-1184 behaves as a cannabinoid receptor antagonist in the guinea-pig myenteric plexus-longitudinal muscle preparation. PMID:9863666

Crystal Structure of the Receptor Binding Domain of the botulinum C-D Mosiac Neurotoxin Reveals Potential Roles of Lysines 1118 and 1136 in Membrane Interactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Y Zhang; G Buchko; L Qin

2011-12-31

The botulinum neurotoxins (BoNTs) produced by different strains of the bacterium Clostridium botulinum are responsible for the disease botulism and include a group of immunologically distinct serotypes (A, B, E, and F) that are considered to be the most lethal natural proteins known for humans. Two BoNT serotypes, C and D, while rarely associated with human infection, are responsible for deadly botulism outbreaks afflicting animals. Also associated with animal infections is the BoNT C-D mosaic protein (BoNT/CD), a BoNT subtype that is essentially a hybrid of the BoNT/C ({approx}two-third) and BoNT/D ({approx}one-third) serotypes. While the amino acid sequence of themore » heavy chain receptor binding (HCR) domain of BoNT/CD (BoNT/CD-HCR) is very similar to the corresponding amino acid sequence of BoNT/D, BoNT/CD-HCR binds synaptosome membranes better than BoNT/D-HCR. To obtain structural insights for the different membrane binding properties, the crystal structure of BoNT/CD-HCR (S867-E1280) was determined at 1.56 {angstrom} resolution and compared to previously reported structures for BoNT/D-HCR. Overall, the BoNT/CD-HCR structure is similar to the two sub-domain organization observed for other BoNT HCRs: an N-terminal jellyroll barrel motif and a C-terminal {beta}-trefoil fold. Comparison of the structure of BoNT/CD-HCR with BoNT/D-HCR indicates that K1118 has a similar structural role as the equivalent residue, E1114, in BoNT/D-HCR, while K1136 has a structurally different role than the equivalent residue, G1132, in BoNT/D-HCR. Lysine-1118 forms a salt bridge with E1247 and may enhance membrane interactions by stabilizing the putative membrane binding loop (K1240-N1248). Lysine-1136 is observed on the surface of the protein. A sulfate ion bound to K1136 may mimic a natural interaction with the negatively changed phospholipid membrane surface. Liposome-binding experiments demonstrate that BoNT/CD-HCR binds phosphatidylethanolamine liposomes more tightly than BoNT/D-HCR.« less

Crystal structure of the receptor binding domain of the botulinum C-D mosaic neurotoxin reveals potential roles of lysines 1118 and 1136 in membrane interactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yanfeng; Buchko, Garry W.; Qin, Ling

2011-01-07

The botulinum neurotoxins (BoNTs) produced by different strains of the bacterium Clostridium botulinum are responsible for the disease botulism and include a group of immunologically distinct serotypes (A, B, E, and F) that are considered to be the most lethal natural proteins known for humans. Two BoNT serotypes, C and D, while rarely associated with human infection, are responsible for deadly botulism outbreaks afflicting animals. Also associated with animal infections is the BoNT C-D mosaic protein (BoNT/CD), a BoNT subtype that is essentially a hybrid of the BoNT/C (~two-thirds) and BoNT/D (~one-third) serotypes. While the amino acid sequence of themore » heavy chain receptor binding (HCR) domain of BoNT/CD (BoNT/CD-HCR) is very similar to the corresponding amino acid sequence of BoNT/D, BoNT/CD-HCR binds synaptosome membranes better than BoNT/D-HCR. To obtain structural insights for the different membrane binding properties, the crystal structure of BoNT/CD-HCR (S867-E1280) was determined at 1.56 Å resolution and compared to previously reported structures for BoNT/D-HCR. Overall, the BoNT/CD-HCR structure is similar to the two sub-domain organization observed for other BoNT HCRs: an N-terminal jellyroll barrel motif and a C-terminal β-trefoil fold. Comparison of the structure of BoNT/CD-HCR with BoNT/D-HCR indicates that K1118 has a similar structural role as the equivalent residue, E1114, in BoNT/D-HCR, while K1136 has a structurally different role than the equivalent residue, G1132, in BoNT/D-HCR. Lysine-1118 forms a salt bridge with E1247 and may enhance membrane interactions by stabilizing the putative membrane binding loop (K1240-N1248). Lysine-1136 is observed on the surface of the protein. A sulfate ion bound to K1136 may mimic a natural interaction with the negatively changed phospholipid membrane surface. Liposome-binding experiments demonstrate that BoNT/CD-HCR binds phosphatidylethanolamine liposomes more tightly than BoNT/D-HCR« less

Crystal structure of the receptor binding domain of the botulinum C-D mosaic neurotoxin reveals potential roles of lysines 1118 and 1136 in membrane interactions.

PubMed

Zhang, Yanfeng; Buchko, Garry W; Qin, Ling; Robinson, Howard; Varnum, Susan M

2011-01-07

The botulinum neurotoxins (BoNTs) produced by different strains of the bacterium Clostridium botulinum are responsible for the disease botulism and include a group of immunologically distinct serotypes (A, B, E, and F) that are considered to be the most lethal natural proteins known for humans. Two BoNT serotypes, C and D, while rarely associated with human infection, are responsible for deadly botulism outbreaks afflicting animals. Also associated with animal infections is the BoNT C-D mosaic protein (BoNT/CD), a BoNT subtype that is essentially a hybrid of the BoNT/C (∼two-third) and BoNT/D (∼one-third) serotypes. While the amino acid sequence of the heavy chain receptor binding (HCR) domain of BoNT/CD (BoNT/CD-HCR) is very similar to the corresponding amino acid sequence of BoNT/D, BoNT/CD-HCR binds synaptosome membranes better than BoNT/D-HCR. To obtain structural insights for the different membrane binding properties, the crystal structure of BoNT/CD-HCR (S867-E1280) was determined at 1.56 Å resolution and compared to previously reported structures for BoNT/D-HCR. Overall, the BoNT/CD-HCR structure is similar to the two sub-domain organization observed for other BoNT HCRs: an N-terminal jellyroll barrel motif and a C-terminal β-trefoil fold. Comparison of the structure of BoNT/CD-HCR with BoNT/D-HCR indicates that K1118 has a similar structural role as the equivalent residue, E1114, in BoNT/D-HCR, while K1136 has a structurally different role than the equivalent residue, G1132, in BoNT/D-HCR. Lysine-1118 forms a salt bridge with E1247 and may enhance membrane interactions by stabilizing the putative membrane binding loop (K1240-N1248). Lysine-1136 is observed on the surface of the protein. A sulfate ion bound to K1136 may mimic a natural interaction with the negatively changed phospholipid membrane surface. Liposome-binding experiments demonstrate that BoNT/CD-HCR binds phosphatidylethanolamine liposomes more tightly than BoNT/D-HCR. Copyright © 2010 Elsevier Inc. All rights reserved.

Ultrasonic isolation of the outer membrane of Escherichia coli with autodisplayed Z-domains.

PubMed

Bong, Ji-Hong; Yoo, Gu; Park, Min; Kang, Min-Jung; Jose, Joachim; Pyun, Jae-Chul

2014-11-01

The outer membrane of Escherichia coli was previously isolated as a liposome-like outer membrane particle using an enzymatic treatment for lysozymes; for immunoassays, the particles were subsequently layered on solid supports via hydrophobic interactions. This work presents an enzyme-free isolation method for the E. coli outer membrane with autodisplayed Z-domains using ultrasonication. First, the properties of the outer membrane particle, such as the particle size, zeta potential, and total protein, were compared with the properties of particles obtained using the previous preparation methods. Compared with the conventional isolation method using an enzyme treatment, the ultrasonic method exhibited a higher efficiency at isolating the outer membrane and less contamination by cytosolic proteins. The isolated outer membrane particles were layered on a gold surface, and the roughness and thickness of the layered outer membrane layers were subsequently analyzed using AFM analysis. Finally, the antibody-binding activity of two outer membrane layers with autodisplayed Z-domains created from particles that were isolated using the enzymatic and ultrasonic isolation methods was measured using fluorescein-labeled antibody as a model analyte, and the activity of the outer membrane layer that was isolated from the ultrasonic method was estimated to be more than 20% higher than that from the conventional enzymatic method. Copyright © 2014 Elsevier Inc. All rights reserved.

Lipid-regulated sterol transfer between closely apposed membranes by oxysterol-binding protein homologues.

PubMed

Schulz, Timothy A; Choi, Mal-Gi; Raychaudhuri, Sumana; Mears, Jason A; Ghirlando, Rodolfo; Hinshaw, Jenny E; Prinz, William A

2009-12-14

Sterols are transferred between cellular membranes by vesicular and poorly understood nonvesicular pathways. Oxysterol-binding protein-related proteins (ORPs) have been implicated in sterol sensing and nonvesicular transport. In this study, we show that yeast ORPs use a novel mechanism that allows regulated sterol transfer between closely apposed membranes, such as organelle contact sites. We find that the core lipid-binding domain found in all ORPs can simultaneously bind two membranes. Using Osh4p/Kes1p as a representative ORP, we show that ORPs have at least two membrane-binding surfaces; one near the mouth of the sterol-binding pocket and a distal site that can bind a second membrane. The distal site is required for the protein to function in cells and, remarkably, regulates the rate at which Osh4p extracts and delivers sterols in a phosphoinositide-dependent manner. Together, these findings suggest a new model of how ORPs could sense and regulate the lipid composition of adjacent membranes.

Elucidation of Lipid Binding Sites on Lung Surfactant Protein A Using X-ray Crystallography, Mutagenesis, and Molecular Dynamics Simulations.

PubMed

Goh, Boon Chong; Wu, Huixing; Rynkiewicz, Michael J; Schulten, Klaus; Seaton, Barbara A; McCormack, Francis X

2016-07-05

Surfactant protein A (SP-A) is a collagenous C-type lectin (collectin) that is critical for pulmonary defense against inhaled microorganisms. Bifunctional avidity of SP-A for pathogen-associated molecular patterns (PAMPs) such as lipid A and for dipalmitoylphosphatidylcholine (DPPC), the major component of surfactant membranes lining the air-liquid interface of the lung, ensures that the protein is poised for first-line interactions with inhaled pathogens. To improve our understanding of the motifs that are required for interactions with microbes and surfactant structures, we explored the role of the tyrosine-rich binding surface on the carbohydrate recognition domain of SP-A in the interaction with DPPC and lipid A using crystallography, site-directed mutagenesis, and molecular dynamics simulations. Critical binding features for DPPC binding include a three-walled tyrosine cage that binds the choline headgroup through cation-π interactions and a positively charged cluster that binds the phosphoryl group. This basic cluster is also critical for binding of lipid A, a bacterial PAMP and target for SP-A. Molecular dynamics simulations further predict that SP-A binds lipid A more tightly than DPPC. These results suggest that the differential binding properties of SP-A favor transfer of the protein from surfactant DPPC to pathogen membranes containing appropriate lipid PAMPs to effect key host defense functions.

Prediction Of pKa From Chemical Structure Using Free And Open-Source Tools

EPA Science Inventory

The ionization state of a chemical, reflected in pKa values, affects lipophilicity, solubility, protein binding and the ability of a chemical to cross the plasma membrane. These properties govern the pharmacokinetic parameters such as absorption, distribution, metabolism, excreti...

2,2'-Bis(monoacylglycero) PO4 (BMP), but Not 3,1'-BMP, increases membrane curvature stress to enhance α-tocopherol transfer protein binding to membranes.

PubMed

Baptist, Matilda; Panagabko, Candace; Nickels, Jonathan D; Katsaras, John; Atkinson, Jeffrey

2015-03-01

Previous work revealed that α-tocopherol transfer protein (α-TTP) co-localizes with bis(monoacylglycero)phosphate (BMP) in late endosomes. BMP is a lipid unique to late endosomes and is believed to induce membrane curvature and support the multivesicular nature of this organelle. We examined the effect of BMP on α-TTP binding to membranes using dual polarization interferometry and vesicle-binding assay. α-TTP binding to membranes is increased by the curvature-inducing lipid BMP. α-TTP binds to membranes with greater affinity when they contain the 2,2'-BMP versus 3,1'-BMP isomers.

2,2'-Bis(monoacylglycero) PO 4 (BMP), but Not 3,1'-BMP, Increases Membrane Curvature Stress to Enhance α-Tocopherol Transfer Protein Binding to Membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baptist, Matilda; Panagabko, Candace; Nickels, Jonathan D.

2015-01-21

Previous work revealed that α-tocopherol transfer protein (α-TTP) co-localizes with bis(monoacylglycero)phosphate (BMP) in late endosomes. BMP is a lipid unique to late endosomes and is believed to induce membrane curvature and support the multivesicular nature of this organelle. In this paper, we examined the effect of BMP on α-TTP binding to membranes using dual polarization interferometry and vesicle-binding assay. α-TTP binding to membranes is increased by the curvature-inducing lipid BMP. Finally, α-TTP binds to membranes with greater affinity when they contain the 2,2'-BMP versus 3,1'-BMP isomers.

Interplay of electrostatics and lipid packing determines the binding of charged polymer coated nanoparticles to model membranes.

PubMed

Biswas, Nupur; Bhattacharya, Rupak; Saha, Arindam; Jana, Nikhil R; Basu, Jaydeep K

2015-10-07

Understanding of nanoparticle-membrane interactions is useful for various applications of nanoparticles like drug delivery and imaging. Here we report on the studies of interaction between hydrophilic charged polymer coated semiconductor quantum dot nanoparticles with model lipid membranes. Atomic force microscopy and X-ray reflectivity measurements suggest that cationic nanoparticles bind and penetrate bilayers of zwitterionic lipids. Penetration and binding depend on the extent of lipid packing and result in the disruption of the lipid bilayer accompanied by enhanced lipid diffusion. On the other hand, anionic nanoparticles show minimal membrane binding although, curiously, their interaction leads to reduction in lipid diffusivity. It is suggested that the enhanced binding of cationic QDs at higher lipid packing can be understood in terms of the effective surface potential of the bilayers which is tunable through membrane lipid packing. Our results bring forth the subtle interplay of membrane lipid packing and electrostatics which determine nanoparticle binding and penetration of model membranes with further implications for real cell membranes.

Drug Distribution. Part 1. Models to Predict Membrane Partitioning.

PubMed

Nagar, Swati; Korzekwa, Ken

2017-03-01

Tissue partitioning is an important component of drug distribution and half-life. Protein binding and lipid partitioning together determine drug distribution. Two structure-based models to predict partitioning into microsomal membranes are presented. An orientation-based model was developed using a membrane template and atom-based relative free energy functions to select drug conformations and orientations for neutral and basic drugs. The resulting model predicts the correct membrane positions for nine compounds tested, and predicts the membrane partitioning for n = 67 drugs with an average fold-error of 2.4. Next, a more facile descriptor-based model was developed for acids, neutrals and bases. This model considers the partitioning of neutral and ionized species at equilibrium, and can predict membrane partitioning with an average fold-error of 2.0 (n = 92 drugs). Together these models suggest that drug orientation is important for membrane partitioning and that membrane partitioning can be well predicted from physicochemical properties.

Using Force to Punch Holes: Mechanics of Contractile Nanomachines.

PubMed

Brackmann, Maximilian; Nazarov, Sergey; Wang, Jing; Basler, Marek

2017-09-01

Using physical force to translocate macromolecules across a membrane has the advantage of being a universal solution independent of the properties of the target membrane. However, physically punching a stiff membrane is not a trivial task and three things are necessary for success: a sharp tip, a source of energy, and the ability to strongly bind to the target. In this review we describe the basic mechanism of membrane puncturing by contractile nanomachines with a focus on the T4 phage, R-type pyocin, and the bacterial Type VI secretion system (T6SS) based on recent studies of the structures and dynamics of their assembly. Copyright © 2017 Elsevier Ltd. All rights reserved.

Role of Endogenous Factors in Response of Erythrocyte Membrane in Patients with Cardiovascular Diseases under Conditions of Ischemic Exposure.

PubMed

Pivovarov, Yu I; Kuznetsova, E E; Koryakina, L B; Gorokhova, V G; Kuril'skaya, T E

2015-05-01

We studied specific features of erythrocyte membrane response to short-term occlusion of the brachial artery in patients with cardiovascular pathology. Under ischemic conditions, processes of sorption were primarily intensified in patients with effort angina and processes of hemoglobin binding with erythrocyte membrane predominated in patients with essential hypertension. These changes in the cell membrane were related to modulation of aggregation properties of erythrocytes (in patients with angina) and plasminogen activity (in patients with essential hypertension). They can also be associated with changes in glucose levels (effort angina) and uric acid (essential hypertension) whose effects can be significantly modified by other endogenous factors.

Oncoprotein AEG-1 is an endoplasmic reticulum RNA-binding protein whose interactome is enriched in organelle resident protein-encoding mRNAs.

PubMed

Hsu, Jack C-C; Reid, David W; Hoffman, Alyson M; Sarkar, Devanand; Nicchitta, Christopher V

2018-05-01

Astrocyte elevated gene-1 (AEG-1), an oncogene whose overexpression promotes tumor cell proliferation, angiogenesis, invasion, and enhanced chemoresistance, is thought to function primarily as a scaffolding protein, regulating PI3K/Akt and Wnt/β-catenin signaling pathways. Here we report that AEG-1 is an endoplasmic reticulum (ER) resident integral membrane RNA-binding protein (RBP). Examination of the AEG-1 RNA interactome by HITS-CLIP and PAR-CLIP methodologies revealed a high enrichment for endomembrane organelle-encoding transcripts, most prominently those encoding ER resident proteins, and within this cohort, for integral membrane protein-encoding RNAs. Cluster mapping of the AEG-1/RNA interaction sites demonstrated a normalized rank order interaction of coding sequence >5' untranslated region, with 3' untranslated region interactions only weakly represented. Intriguingly, AEG-1/membrane protein mRNA interaction sites clustered downstream from encoded transmembrane domains, suggestive of a role in membrane protein biogenesis. Secretory and cytosolic protein-encoding mRNAs were also represented in the AEG-1 RNA interactome, with the latter category notably enriched in genes functioning in mRNA localization, translational regulation, and RNA quality control. Bioinformatic analyses of RNA-binding motifs and predicted secondary structure characteristics indicate that AEG-1 lacks established RNA-binding sites though shares the property of high intrinsic disorder commonly seen in RBPs. These data implicate AEG-1 in the localization and regulation of secretory and membrane protein-encoding mRNAs and provide a framework for understanding AEG-1 function in health and disease. © 2018 Hsu et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Pharmacological analysis of [3H]-senktide binding to NK3 tachykinin receptors in guinea-pig ileum longitudinal muscle-myenteric plexus and cerebral cortex membranes.

PubMed Central

Guard, S.; Watson, S. P.; Maggio, J. E.; Too, H. P.; Watling, K. J.

1990-01-01

1. The binding properties and pharmacological specificity of the selective NK3 tachykinin receptor agonist [3H))-senktide [( 3H]-succinyl[Asp6,MePhe8] substance P (6-11] have been examined in homogenates of guinea-pig ileum longitudinal muscle-myenteric plexus (LM/MP) and cerebral cortex. 2. Scatchard analysis of saturation binding studies in guinea-pig ileum LM/MP and cerebral cortex membranes indicated that [3H]-senktide bound to a single site with apparent high affinity, KD = 2.21 +/- 0.65 nM; Bmax = 13.49 +/- 0.04 fmol mg-1 protein in ileum and KD = 8.52 +/- 0.45 nM; Bmax = 76.3 +/- 1.6 fmol mg-1 protein in cortex (values are means +/- ranges; n = 2). 3. The pharmacological profile for tachykinins and analogues in displacing [3H]-senktide from ileum membranes was: [MePhe7] neurokinin B greater than neurokinin B (NKB) congruent to senktide greater than eledoisin greater than substance P (SP) greater than neurokinin A(NKA) greater than physalaemin greater than [Sar9,Met(O2)11]SP greater than [Nle10]NKA(4-10) = [Glp6,L-Pro9]-SP(6-11) greater than substance P methyl ester, consistent with [3H]-senktide binding to an NK3 subtype of tachykinin receptor. A similar rank order of affinity was obtained for these peptides in displacing [3H]-senktide from cortex membranes. 4. Several tachykinin receptor agonists were tested for their ability to displace [3H]-senktide from ileal and cortical NK3 binding sites and were found to be either weak displacers (pIC50 less than 5.00) or inactive.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1694464

The two envelope membrane glycoproteins of Tomato spotted wilt virus show differences in lectin-binding properties and sensitivities to glycosidases.

PubMed

Naidu, Rayapati A; Ingle, Caroline J; Deom, Carl M; Sherwood, John L

2004-02-05

Tomato spotted wilt virus (TSWV, Genus: Tospovirus, Family: Bunyaviridae) is a major constraint to the production of several different crops of agronomic and horticultural importance worldwide. The amino acid sequence of the two envelope membrane glycoproteins, designated as G(N) (N-terminal) and G(C) (C-terminal), of TSWV contain several tripeptide sequences, Asn-Xaa-Ser/Thr, suggesting that the proteins are N-glycosylated. In this study, the lectin-binding properties of the viral glycoproteins and their sensitivities to glycosidases were examined to obtain information on the nature of potential oligosaccharide moieties present on G(N) and G(C). The viral proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and probed by affinoblotting using a battery of biotinylated lectins with specificity to different oligosaccharide structures. G(C) showed strong binding with five mannose-binding lectins, four N-acetyllactosamine-binding lectins and one fucose-binding lectin. G(N) was resolved into two molecular masses and only the slow migrating form showed binding, albeit to a lesser extent than G(C), with three of the five mannose-binding lectins. The N-acetyllactosamine- and fucose-specific lectins did not bind to either molecular mass form of G(N). None of the galactose-, N-acetylgalactosamine-, or sialic acid-binding lectins tested showed binding specificity to G(C) or G(N). Treatment of the denatured virions with endoglycosidase H and peptide:N-glycosidase F (PNGase F) resulted in a significant decrease in the binding of G(C) to high mannose- and N-acetyllactosamine-specific lectins. However, no such differences in lectin binding were apparent with G(N). These results indicate the presence of N-linked oligosaccharides of high mannose- and complex-type on G(C) and possibly high mannose-type on G(N). Differences in the extent of binding of the two envelope glycoproteins to different lectins suggest that G(C) is likely to be more heavily N-glycosylated than G(N). No evidence was observed for the presence of O-linked oligosaccharides on G(N) or G(C).

Advances in immobilized artificial membrane (IAM) chromatography for novel drug discovery.

PubMed

Tsopelas, Fotios; Vallianatou, Theodosia; Tsantili-Kakoulidou, Anna

2016-01-01

The development of immobilized artificial membrane (IAM) chromatography has unfolded new perspectives for the use of chromatographic techniques in drug discovery, combining simulation of the environment of cell membranes with rapid measurements. The present review describes the characteristics of phosphatidylcholine-based stationary phases and analyses the molecular factors governing IAM retention in comparison to n-octanol-water and liposomes partitioning systems as well as to reversed phase chromatography. Other biomimetic stationary phases are also briefly discussed. The potential of IAM chromatography to model permeability through the main physiological barriers and drug membrane interactions is outlined. Further applications to calculate complex pharmacokinetic properties, related to tissue binding, and to screen drug candidates for phospholipidosis, as well as to estimate cell accumulation/retention are surveyed. The ambivalent nature of IAM chromatography, as a border case between passive diffusion and binding, defines its multiple potential applications. However, despite its successful performance in many permeability and drug-membrane interactions studies, IAM chromatography is still used as a supportive and not a stand-alone technique. Further studies looking at IAM chromatography in different biological processes are still required if this technique is to have a more focused and consistent application in drug discovery.

Attack on single Escherichia coli spheroplast by antimicrobial peptides

NASA Astrophysics Data System (ADS)

Sun, Tzu-Lin

Studies of the molecular mechanisms of antimicrobial peptides (AMPs) have mostly been performed with lipid bilayers, as a substitute for cell membrane. Hence, there is a persistent question as to whether the action of AMPs on bacterial membranes can be reproduced on lipid bilayers. Valuable information was obtained recently from observing the actions of AMPs on E. coli and Bacillus subtilis by time-lapse fluorescence microscopy. The goal of my dissertation is to study the direct action of AMPs on the cytoplasmic membranes by using E. coli spheroplasts, the cell form from which the outer membranes have been removed. The key question is how to reveal the response of the spheroplast to AMPs. In our previous work, the aspiration method on giant unilamellar vesicle (GUV) has been demonstrated as very effective for researching membrane effects induced by peptides. This method is able to measure the membrane expansion due to peptide binding and simultaneously monitor membrane permeability by using dye indicators. In this work, a spray method was developed for introducing AMPs and customized the experimental procedures for performing the experiments of E. coli spheroplasts. The living state of cytoplasmic membrane makes it difficult to deduce the molecular events from the response of live cells. Hence, the physical methods which have been developed for researching peptide activity on lipid bilayers were practiced first. These methods include X-ray diffraction (XRD), oriented circular dichroism (OCD) and GUV aspiration. These methods were practiced by studying the question as to why a hydrocarbon-stapled peptide NYAD-1 drug was reported to have membrane permeating property. Melittin was selected as the representative of AMPs for the E. coli spheroplast experiments. To better understand the characteristics of melittin, the melittin activity on model lipid membrane was examined before advancing to the spehroplast experiment. The melittin transmembrane was proposed by correlating melittin binding on a lipid vesicle (aspirated GUV imaging) with structural studies in multilayers (XRD and OCD). This behavior was further determined by using fluorescence indicator to track the melittin distribution. The toroidal structure of melittin pore was also detected by grazing-angle X-ray anomalous diffraction. In the studies of NAYD-1 and melittin on a model membrane, our discovery of AMP's transmembrane enables us to clarify the pore-formation mechanism which has been disputed for decades. In addition, our past work on the physical property of E. coli spheroplast cytoplasmic membrane has indicated the existence of a lipid reservoir. The lipid reservoir dominates the surface tension by balancing the membrane folds. Finally, we used the aspiration method to hold a spheroplast so as to measure the change of the spheroplast membrane area in response to the AMPs' binding. Further, a fluorescence indicator which is able to associate with AMPs was used to monitor the peptide distribution and another fluorescence dye to monitor the molecule leakage. The spheroplast study shows that there are similarities and differences between the responses of spheroplasts and GUVs. The recent findings on the unique properties of spheroplast membranes are the key for understanding these results. Our work of understanding the AMPs activity on membrane is potentially applicable in improving peptide drug design and delivery for disease treatment.

20180318 - Prediction Of pKa From Chemical Structure Using Free And Open-Source Tools (ACS Spring)

EPA Science Inventory

The ionization state of a chemical, reflected in pKa values, affects lipophilicity, solubility, protein binding and the ability of a chemical to cross the plasma membrane. These properties govern the pharmacokinetic parameters such as absorption, distribution, metabolism, excreti...

Characterization of bradykinin receptors in human lung fibroblasts using the binding of 3[H][Des-Arg10,Leu9]kallidin and [3H]NPC17731.

PubMed

Zhang, S P; Codd, E E

1998-01-01

Bradykinin (BK) receptors are involved in pain and inflammation. Two BK receptor subtypes, B1 and B2, have been defined based on their pharmacological properties. Both B1 and B2 receptors are G-protein coupled membrane receptors. B1 receptors are present in smooth muscle tissue, whereas B2 receptors are found in both smooth muscle tissue and neurons. [Des-Arg10,Leu9]kallidin (DALKD) is a selective B1 receptor antagonist, and NPC17731 is a selective B2 receptor antagonist. To develop binding assays for the two known BK receptor subtypes, [3H]DALKD and [3H]NPC17731 were used as selective ligands for B1 and B2 receptors respectively. Both ligands bound to the CCD-16 human lung fibroblast membranes reaching equilibrium at 25 degrees C within 30 min. Binding was stable for at least 60 min. The Kd of [3H]DALKD was 0.33 nM and Bmax was 52 fmol/mg membrane protein. The Kd of [3H]NPC17731 was 0.39 nM and Bmax was 700 fmol/mg membrane protein. Competition for [3H]DALKD binding with BK receptor agonists was in the order: [des-Arg10]KD (DAKD) > KD > [des-Arg9]BK (DABK) > BK, and competition for [3H]DALKD binding with BK receptor antagonists was in the order: DALKD > [des-Arg10]Hoe 140 (DAHoe 140) > [des-Arg9,Leu8]BK (DALBK) > NPC17731 > Hoe 140 > DNMFBK, suggesting that [3H]DALKD bound selectively to B1 receptors. By contrast, competition for [3H]NPC17731 binding by BK agonists was in the order: BK > KD > DAKD > DABK, and competition for [3H]NPC17731 binding by BK antagonists was in the order: NPC17731 = Hoe 140 > DNMFBK > DAHoe 140 > DALBK > DALKD, indicating that [3H]NPC17731 labeled B2 receptors selectively. These results demonstrate that [3H]DALKD and [3H]NPC17731 can be used with CCD-16 human lung fibroblast membranes to provide a pair of binding assays for the simultaneous evaluation of B1 and B2 BK receptor subtypes.

Two interdependent mechanisms of antimicrobial activity allow for efficient killing in nylon-3-based polymeric mimics of innate immunity peptides ☆

PubMed Central

Lee, Michelle W.; Chakraborty, Saswata; Schmidt, Nathan W.; Murgai, Rajan; Gellman, Samuel H.; Wong, Gerard C.L.

2015-01-01

Novel synthetic mimics of antimicrobial peptides have been developed to exhibit structural properties and antimicrobial activity similar to those of natural antimicrobial peptides (AMPs) of the innate immune system. These molecules have a number of potential advantages over conventional antibiotics, including reduced bacterial resistance, cost-effective preparation, and customizable designs. In this study, we investigate a family of nylon-3 polymer-based antimicrobials. By combining vesicle dye leakage, bacterial permeation, and bactericidal assays with small-angle X-ray scattering (SAXS), we find that these polymers are capable of two interdependent mechanisms of action: permeation of bacterial membranes and binding to intracellular targets such as DNA, with the latter necessarily dependent on the former. We systemically examine polymer-induced membrane deformation modes across a range of lipid compositions that mimic both bacteria and mammalian cell membranes. The results show that the polymers' ability to generate negative Gaussian curvature (NGC), a topological requirement for membrane permeation and cellular entry, in model Escherichia coli membranes correlates with their ability to permeate membranes without complete membrane disruption and kill E. coli cells. Our findings suggest that these polymers operate with a concentration dependent mechanism of action: at low concentrations permeation and DNA binding occur without membrane disruption, while at high concentrations complete disruption of the membrane occurs. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. PMID:24743021

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yee, J.P.

The following studies were conducted using the resistive pulse spectroscopy (RPS) technique: cumulative spectra and individual pulse forms for rigid latex polymer spheres; acquisition and analysis of RPS spectral data by means of special computer program; interaction of red blood cells with glutaraldehyde; membrane properties of erythrocytes undergoing abrupt osmotic hemolysis; reversible effects of the binding of chlorpromazine HCl at the red cell membrane surface; effects of high cholesterol diet on erythrocytes of guinea pigs; and multi-population analysis for a mixture of fetal and maternal red cells. (HLW)

Size-dependent protein segregation at membrane interfaces

NASA Astrophysics Data System (ADS)

Schmid, Eva M.; Bakalar, Matthew H.; Choudhuri, Kaushik; Weichsel, Julian; Ann, Hyoung Sook; Geissler, Phillip L.; Dustin, Michael L.; Fletcher, Daniel A.

2016-07-01

Membrane interfaces formed at cell-cell junctions are associated with characteristic patterns of membrane proteins whose organization is critical for intracellular signalling. To isolate the role of membrane protein size in pattern formation, we reconstituted model membrane interfaces in vitro using giant unilamellar vesicles decorated with synthetic binding and non-binding proteins. We show that size differences between membrane proteins can drastically alter their organization at membrane interfaces, with as little as a ~5 nm increase in non-binding protein size driving its exclusion from the interface. Combining in vitro measurements with Monte Carlo simulations, we find that non-binding protein exclusion is also influenced by lateral crowding, binding protein affinity, and thermally driven membrane height fluctuations that transiently limit access to the interface. This sensitive and highly effective means of physically segregating proteins has implications for cell-cell contacts such as T-cell immunological synapses (for example, CD45 exclusion) and epithelial cell junctions (for example, E-cadherin enrichment), as well as for protein sorting at intracellular contact points between membrane-bound organelles.

Size-dependent protein segregation at membrane interfaces

PubMed Central

Schmid, Eva M; Bakalar, Matthew H; Choudhuri, Kaushik; Weichsel, Julian; Ann, HyoungSook; Geissler, Phillip L; Dustin, Michael L; Fletcher, Daniel A

2016-01-01

Membrane interfaces formed at cell-cell junctions are associated with characteristic patterns of membrane protein organization, such as E-cadherin enrichment in epithelial junctional complexes and CD45 exclusion from the signaling foci of immunological synapses. To isolate the role of protein size in these processes, we reconstituted membrane interfaces in vitro using giant unilamellar vesicles decorated with synthetic binding and non-binding proteins. We show that size differences between binding and non-binding proteins can dramatically alter their organization at membrane interfaces in the absence of active contributions from the cytoskeleton, with as little as a ~5 nm increase in non-binding protein size driving its exclusion from the interface. Combining in vitro measurements with Monte Carlo simulations, we find that non-binding protein exclusion is also influenced by lateral crowding, binding protein affinity, and thermally-driven membrane height fluctuations that transiently limit access to the interface. This simple, sensitive, and highly effective means of passively segregating proteins has implications for signaling at cell-cell junctions and protein sorting at intracellular contact points between membrane-bound organelles. PMID:27980602

Hemagglutinin-Mediated Membrane Fusion: A Biophysical Perspective.

PubMed

Boonstra, Sander; Blijleven, Jelle S; Roos, Wouter H; Onck, Patrick R; van der Giessen, Erik; van Oijen, Antoine M

2018-05-20

Influenza hemagglutinin (HA) is a viral membrane protein responsible for the initial steps of the entry of influenza virus into the host cell. It mediates binding of the virus particle to the host-cell membrane and catalyzes fusion of the viral membrane with that of the host. HA is therefore a major target in the development of antiviral strategies. The fusion of two membranes involves high activation barriers and proceeds through several intermediate states. Here, we provide a biophysical description of the membrane fusion process, relating its kinetic and thermodynamic properties to the large conformational changes taking place in HA and placing these in the context of multiple HA proteins working together to mediate fusion. Furthermore, we highlight the role of novel single-particle experiments and computational approaches in understanding the fusion process and their complementarity with other biophysical approaches.

Force Spectroscopy Reveals the Effect of Different Ions in the Nanomechanical Behavior of Phospholipid Model Membranes: The Case of Potassium Cation

PubMed Central

Redondo-Morata, Lorena; Oncins, Gerard; Sanz, Fausto

2012-01-01

How do metal cations affect the stability and structure of phospholipid bilayers? What role does ion binding play in the insertion of proteins and the overall mechanical stability of biological membranes? Investigators have used different theoretical and microscopic approaches to study the mechanical properties of lipid bilayers. Although they are crucial for such studies, molecular-dynamics simulations cannot yet span the complexity of biological membranes. In addition, there are still some experimental difficulties when it comes to testing the ion binding to lipid bilayers in an accurate way. Hence, there is a need to establish a new approach from the perspective of the nanometric scale, where most of the specific molecular phenomena take place. Atomic force microscopy has become an essential tool for examining the structure and behavior of lipid bilayers. In this work, we used force spectroscopy to quantitatively characterize nanomechanical resistance as a function of the electrolyte composition by means of a reliable molecular fingerprint that reveals itself as a repetitive jump in the approaching force curve. By systematically probing a set of bilayers of different composition immersed in electrolytes composed of a variety of monovalent and divalent metal cations, we were able to obtain a wealth of information showing that each ion makes an independent and important contribution to the gross mechanical resistance and its plastic properties. This work addresses the need to assess the effects of different ions on the structure of phospholipid membranes, and opens new avenues for characterizing the (nano)mechanical stability of membranes. PMID:22225799

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beaumont, K.; Vaughn, D.A.; Fanestil, D.D.

Thiazides and related diuretics inhibit NaCl reabsorption in the distal tubule through an unknown mechanism. The authors report here that ({sup 3}H)metolazone, a diuretic with a thiazide-like mechanism of action, labels a site in rat kidney membranes that has characteristics of the thiazide-sensitive ion transporter. ({sup 3}H)Metolazone bound with high affinity to a site with a density of 0.717 pmol/mg of protein in kidney membranes. The binding site was localized to the renal cortex, with little or not binding in other kidney regions and 11 other tissues. The affinities of thiazide-type diuretics for this binding site were significantly correlated withmore » their clinical potency. Halide anions specifically inhibited high-affinity binding of ({sup 3}H)metolazone to this site. ({sup 3})Metolazone also bound with lower affinity to sites present in kidney as well as in liver, testis, lung, brain, heart, and other tissues. Calcium antagonists and certain smooth muscle relaxants had K{sub i} values of 0.6-10 {mu}M for these low-affinity sites, which were not inhibited by most of the thiazide diuretics tested. Properties of the high-affinity ({sup 3}H)metolazone binding site are consistent with its identity as the receptor for thiazide-type diuretics.« less

Leveraging the membrane-cytoskeleton interface with myosin-1

PubMed Central

McConnell, Russell E.; Tyska, Matthew J.

2010-01-01

Class 1 myosins are small motor proteins with the ability to simultaneously bind to actin filaments and cellular membranes. Given their ability to generate mechanical force, and their high prevalence in many cell types, these molecules are well positioned to carry out a number of important biological functions at the interface of membrane and the actin cytoskeleton. Indeed, recent studies implicate these motors in endocytosis, exocytosis, release of extracellular vesicles, and the regulation of tension between membrane and the cytoskeleton. Many class 1 myosins also exhibit a load-dependent mechano-chemical cycle that enables them to maintain tension for long periods of time without hydrolyzing ATP. These properties put myosins-1 in a unique position to regulate dynamic membrane-cytoskeleton interactions and respond to physical forces during these events. PMID:20471271

Molecular Dynamics Simulations of Creatine Kinase and Adenine Nucleotide Translocase in Mitochondrial Membrane Patch*

PubMed Central

Karo, Jaanus; Peterson, Pearu; Vendelin, Marko

2012-01-01

Interaction between mitochondrial creatine kinase (MtCK) and adenine nucleotide translocase (ANT) can play an important role in determining energy transfer pathways in the cell. Although the functional coupling between MtCK and ANT has been demonstrated, the precise mechanism of the coupling is not clear. To study the details of the coupling, we turned to molecular dynamics simulations. We introduce a new coarse-grained molecular dynamics model of a patch of the mitochondrial inner membrane containing a transmembrane ANT and an MtCK above the membrane. The membrane model consists of three major types of lipids (phosphatidylcholine, phosphatidylethanolamine, and cardiolipin) in a roughly 2:1:1 molar ratio. A thermodynamics-based coarse-grained force field, termed MARTINI, has been used together with the GROMACS molecular dynamics package for all simulated systems in this work. Several physical properties of the system are reproduced by the model and are in agreement with known data. This includes membrane thickness, dimension of the proteins, and diffusion constants. We have studied the binding of MtCK to the membrane and demonstrated the effect of cardiolipin on the stabilization of the binding. In addition, our simulations predict which part of the MtCK protein sequence interacts with the membrane. Taken together, the model has been verified by dynamical and structural data and can be used as the basis for further studies. PMID:22241474

Receptor binding properties and antinociceptive effects of chimeric peptides consisting of a micro-opioid receptor agonist and an ORL1 receptor antagonist.

PubMed

Kawano, Susumu; Ito, Risa; Nishiyama, Miharu; Kubo, Mai; Matsushima, Tomoko; Minamisawa, Motoko; Ambo, Akihiro; Sasaki, Yusuke

2007-07-01

Receptor binding properties and antinociceptive activities of chimeric peptides linked by spacers were investigated. The peptides consisted of the micro-opioid receptor ligand dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH(2)) or its analog YRFB (Tyr-D-Arg-Phe-betaAla-NH(2)) linked to the ORL1 receptor ligand Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-NH(2) (Ac-RYYRIK-NH(2)). All chimeric peptides were found to possess high receptor binding affinities for both micro-opioid and ORL1 receptors in mouse brain membranes although their binding affinities for both receptors in spinal membranes were significantly lower. Among them, chimeric peptide 2, which consists of dermorphin and Ac-RYYRIK-NH(2) connected by a long spacer, had the highest binding affinity towards both receptors. In the tail-flick test following intrathecal (i.t.) administration to mice, all chimeric peptides showed potent and dose-dependent antinociceptive activities with an ED(50) of 1.34-4.51 (pmol/mouse), nearly comparable to dermorphin alone (ED(50); 1.08 pmol/mouse). In contrast to their micro-opioid receptor binding profiles, intracerebroventricular (i.c.v.) administration of the chimeric peptides resulted in much less potent antinociceptive activity (ED(50) 5.55-100< pmol/mouse) than when administered i.t. (ED(50): 1.34-4.51 pmol/mouse). These results suggest the involvement of nociceptin-like agonistic effects of the Ac-RYYRIK pharmacophore in the peptides, and the regulation of mu-opioid receptor-mediated antinociception in brain. The present chimeric peptides may be useful as pharmacological tools for studies on micro-opioid receptor/ORL1 receptor heterodimers.

Binding of Signal Recognition Particle Gives Ribosome/Nascent Chain Complexes a Competitive Advantage in Endoplasmic Reticulum Membrane Interaction

PubMed Central

Neuhof, Andrea; Rolls, Melissa M.; Jungnickel, Berit; Kalies, Kai-Uwe; Rapoport, Tom A.

1998-01-01

Most secretory and membrane proteins are sorted by signal sequences to the endoplasmic reticulum (ER) membrane early during their synthesis. Targeting of the ribosome-nascent chain complex (RNC) involves the binding of the signal sequence to the signal recognition particle (SRP), followed by an interaction of ribosome-bound SRP with the SRP receptor. However, ribosomes can also independently bind to the ER translocation channel formed by the Sec61p complex. To explain the specificity of membrane targeting, it has therefore been proposed that nascent polypeptide-associated complex functions as a cytosolic inhibitor of signal sequence- and SRP-independent ribosome binding to the ER membrane. We report here that SRP-independent binding of RNCs to the ER membrane can occur in the presence of all cytosolic factors, including nascent polypeptide-associated complex. Nontranslating ribosomes competitively inhibit SRP-independent membrane binding of RNCs but have no effect when SRP is bound to the RNCs. The protective effect of SRP against ribosome competition depends on a functional signal sequence in the nascent chain and is also observed with reconstituted proteoliposomes containing only the Sec61p complex and the SRP receptor. We conclude that cytosolic factors do not prevent the membrane binding of ribosomes. Instead, specific ribosome targeting to the Sec61p complex is provided by the binding of SRP to RNCs, followed by an interaction with the SRP receptor, which gives RNC–SRP complexes a selective advantage in membrane targeting over nontranslating ribosomes. PMID:9436994

Membrane Destruction and DNA Binding of Staphylococcus aureus Cells Induced by Carvacrol and Its Combined Effect with a Pulsed Electric Field.

PubMed

Wang, Lang-Hong; Wang, Man-Sheng; Zeng, Xin-An; Zhang, Zhi-Hong; Gong, De-Ming; Huang, Yan-Bo

2016-08-17

Carvacrol (5-isopropyl-2-methylphenol, CAR) is an antibacterial ingredient that occurs naturally in the leaves of the plant Origanum vulgare. The antimicrobial mechanism of CAR against Staphylococcus aureus ATCC 43300 was investigated in the study. Analysis of the membrane fatty acids by gas chromatography-mass spectrometry (GC-MS) showed that exposure to CAR at low concentrations induced a marked increase in the level of unbranched fatty acids (from 34.90 ± 1.77% to 62.37 ± 4.26%). Moreover, CAR at higher levels severely damaged the integrity and morphologies of the S. aureus cell membrane. The DNA-binding properties of CAR were also investigated using fluorescence, circular dichroism, molecular modeling, and atomic-force microscopy. The results showed that CAR bound to DNA via the minor-groove mode, mildly perturbed the DNA secondary structure, and induced DNA molecules to be aggregated. Furthermore, a combination of CAR with a pulsed-electric field was found to exhibit strong synergistic effects on S. aureus.

The rhodopsin-arrestin-1 interaction in bicelles.

PubMed

Chen, Qiuyan; Vishnivetskiy, Sergey A; Zhuang, Tiandi; Cho, Min-Kyu; Thaker, Tarjani M; Sanders, Charles R; Gurevich, Vsevolod V; Iverson, T M

2015-01-01

G-protein-coupled receptors (GPCRs) are essential mediators of information transfer in eukaryotic cells. Interactions between GPCRs and their binding partners modulate the signaling process. For example, the interaction between GPCR and cognate G protein initiates the signal, while the interaction with cognate arrestin terminates G-protein-mediated signaling. In visual signal transduction, arrestin-1 selectively binds to the phosphorylated light-activated GPCR rhodopsin to terminate rhodopsin signaling. Under physiological conditions, the rhodopsin-arrestin-1 interaction occurs in highly specialized disk membrane in which rhodopsin resides. This membrane is replaced with mimetics when working with purified proteins. While detergents are commonly used as membrane mimetics, most detergents denature arrestin-1, preventing biochemical studies of this interaction. In contrast, bicelles provide a suitable alternative medium. An advantage of bicelles is that they contain lipids, which have been shown to be necessary for normal rhodopsin-arrestin-1 interaction. Here we describe how to reconstitute rhodopsin into bicelles, and how bicelle properties affect the rhodopsin-arrestin-1 interaction.

The Rhodopsin-Arrestin-1 Interaction in Bicelles

PubMed Central

Chen, Qiuyan; Vishnivetskiy, Sergey A.; Zhuang, Tiandi; Cho, Min-Kyu; Thaker, Tarjani M.; Sanders, Charles R.; Gurevich, Vsevolod V.; Iverson, T. M.

2015-01-01

G-protein-coupled receptors (GPCRs) are essential mediators of information transfer in eukaryotic cells. Interactions between GPCRs and their binding partners modulate the signaling process. For example, the interaction between GPCR and cognate G protein initiates the signal, while the interaction with cognate arrestin terminates G-protein-mediated signaling. In visual signal transduction, arrestin-1 selectively binds to the phosphorylated light-activated GPCR rhodopsin to terminate rhodopsin signaling. Under physiological conditions, the rhodopsin-arrestin-1 interaction occurs in highly specialized disk membrane in which rhodopsin resides. This membrane is replaced with mimetics when working with purified proteins. While detergents are commonly used as membrane mimetics, most detergents denature arrestin-1, preventing biochemical studies of this interaction. In contrast, bicelles provide a suitable alternative medium. An advantage of bicelles is that they contain lipids, which have been shown to be necessary for normal rhodopsin-arrestin-1 interaction. Here we describe how to reconstitute rhodopsin into bicelles, and how bicelle properties affect the rhodopsin-arrestin-1 interaction. PMID:25697518

Effects of sterol-binding agent nystatin on wheat roots: the changes in membrane permeability, sterols and glycoceramides.

PubMed

Valitova, Julia N; Minibayeva, Farida V; Kotlova, Ekaterina R; Novikov, Alexander V; Shavarda, Alexey L; Murtazina, Lyaisan I; Ryzhkina, Irina S

2011-10-01

Plant sterols are important multifunctional lipids, which are involved in determining membrane properties. Biophysical characteristics of model lipid and isolated animal membranes with altered sterol component have been intensively studied. In plants however, the precise mechanisms of involvement of sterols in membrane functioning remain unclear. In present work the possible interactions between sterols and other membrane lipids in plant cells were studied. A useful experimental approach for elucidating the roles of sterols in membrane activity is to use agents that specifically bind with endogenous sterols, for example the antibiotic nystatin. Membrane characteristics and the composition of membrane lipids in the roots of wheat (Triticum aestivum L.) seedlings treated with nystatin were analyzed. The application of nystatin greatly increased the permeability of the plasma membrane for ions and SH-containing molecules and decreased the total sterol level mainly as a consequence of a reduction in the amount of β-sitosterol and campesterol. Dynamic light-scattering was used to confirm the in vitro formation of stable complexes between nystatin and β-sitosterol or cholesterol. Sterol depletion was accompanied by a significant rise in total glycoceramide (GlCer) content after 2h treatment with nystatin. Analysis of the GlCer composition using mass spectrometry with electrospray ionization demonstrated that nystatin induced changes in the ratio of molecular species of GlCer. Our results suggest that changes in the sphingolipid composition can contribute to the changes in plasma membrane functioning induced by sterol depletion. Copyright © 2011 Elsevier Ltd. All rights reserved.

Myosin IIA interacts with the spectrin-actin membrane skeleton to control red blood cell membrane curvature and deformability.

PubMed

Smith, Alyson S; Nowak, Roberta B; Zhou, Sitong; Giannetto, Michael; Gokhin, David S; Papoin, Julien; Ghiran, Ionita C; Blanc, Lionel; Wan, Jiandi; Fowler, Velia M

2018-05-08

The biconcave disk shape and deformability of mammalian RBCs rely on the membrane skeleton, a viscoelastic network of short, membrane-associated actin filaments (F-actin) cross-linked by long, flexible spectrin tetramers. Nonmuscle myosin II (NMII) motors exert force on diverse F-actin networks to control cell shapes, but a function for NMII contractility in the 2D spectrin-F-actin network of RBCs has not been tested. Here, we show that RBCs contain membrane skeleton-associated NMIIA puncta, identified as bipolar filaments by superresolution fluorescence microscopy. MgATP disrupts NMIIA association with the membrane skeleton, consistent with NMIIA motor domains binding to membrane skeleton F-actin and contributing to membrane mechanical properties. In addition, the phosphorylation of the RBC NMIIA heavy and light chains in vivo indicates active regulation of NMIIA motor activity and filament assembly, while reduced heavy chain phosphorylation of membrane skeleton-associated NMIIA indicates assembly of stable filaments at the membrane. Treatment of RBCs with blebbistatin, an inhibitor of NMII motor activity, decreases the number of NMIIA filaments associated with the membrane and enhances local, nanoscale membrane oscillations, suggesting decreased membrane tension. Blebbistatin-treated RBCs also exhibit elongated shapes, loss of membrane curvature, and enhanced deformability, indicating a role for NMIIA contractility in promoting membrane stiffness and maintaining RBC biconcave disk cell shape. As structures similar to the RBC membrane skeleton exist in many metazoan cell types, these data demonstrate a general function for NMII in controlling specialized membrane morphology and mechanical properties through contractile interactions with short F-actin in spectrin-F-actin networks.

How synthetic membrane systems contribute to the understanding of lipid-driven endocytosis.

PubMed

Schubert, Thomas; Römer, Winfried

2015-11-01

Synthetic membrane systems, such as giant unilamellar vesicles and solid supported lipid bilayers, have widened our understanding of biological processes occurring at or through membranes. Artificial systems are particularly suited to study the inherent properties of membranes with regard to their components and characteristics. This review critically reflects the emerging molecular mechanism of lipid-driven endocytosis and the impact of model membrane systems in elucidating the complex interplay of biomolecules within this process. Lipid receptor clustering induced by binding of several toxins, viruses and bacteria to the plasma membrane leads to local membrane bending and formation of tubular membrane invaginations. Here, lipid shape, and protein structure and valency are the essential parameters in membrane deformation. Combining observations of complex cellular processes and their reconstitution on minimal systems seems to be a promising future approach to resolve basic underlying mechanisms. This article is part of a Special Issue entitled: Mechanobiology. Copyright © 2015 Elsevier B.V. All rights reserved.

[Mechanism of action of neurotoxins acting on the inactivation of voltage-gated sodium channels].

PubMed

Benoit, E

1998-01-01

This review focuses on the mechanism(s) of action of neurotoxins acting on the inactivation of voltage-gated Na channels. Na channels are transmembrane proteins which are fundamental for cellular communication. These proteins form pores in the plasma membrane allowing passive ionic movements to occur. Their opening and closing are controlled by gating systems which depend on both membrane potential and time. Na channels have three functional properties, mainly studied using electrophysiological and biochemical techniques, to ensure their role in the generation and propagation of action potentials: 1) a highly selectivity for Na ions, 2) a rapid opening ("activation"), responsible for the depolarizing phase of the action potential, and 3) a late closing ("inactivation") involved in the repolarizing phase of the action potential. As an essential protein for membrane excitability, the Na channel is the specific target of a number of vegetal and animal toxins which, by binding to the channel, alter its activity by affecting one or more of its properties. At least six toxin receptor sites have been identified on the neuronal Na channel on the basis of binding studies. However, only toxins interacting with four of these sites (sites 2, 3, 5 et 6) produce alterations of channel inactivation. The maximal percentage of Na channels modified by the binding of neurotoxins to sites 2 (batrachotoxin and some alkaloids), 3 (alpha-scorpion and sea anemone toxins), 5 (brevetoxins and ciguatoxins) et 6 (delta-conotoxins) is different according to the site considered. However, in all cases, these channels do not inactivate. Moreover, Na channels modified by toxins which bind to sites 2, 5 and 6 activate at membrane potentials more negative than do unmodified channels. The physiological consequences of Na channel modifications, induced by the binding of neurotoxins to sites 2, 3, 5 and 6, are (i) an inhibition of cellular excitability due to an important membrane depolarization (site 2), (ii) a decrease of cellular excitability due to an important increase in the action potential duration (site 3) and (iii) an increase in cellular excitability which results in spontaneous and repetitive firing of action potentials (sites 5 and 6). The biochemical and electrophysiological studies performed with these toxins, as well as the determination of their molecular structure, have given basic information on the function and structure of the Na channel protein. Therefore, various models representing the different states of Na channels have been proposed to account for the neurotoxin-induced modifications of Na inactivation. Moreover, the localization of receptor binding sites 2, 3, 5 et 6 for these toxins on the neuronal Na channel has been deduced and the molecular identification of the recognition site(s) for some of them has been established on the alpha sub-unit forming the Na channel protein.

Binding of (/sup 3/H)forskolin to solubilized preparations of adenylate cyclase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nelson, C.A.; Seamon, K.B.

1988-01-01

The binding of (/sup 3/H)forskolin to proteins solubilized from bovine brain membranes was studied by precipitating proteins with polyethylene glycol and separating (/sup 3/H)forskolin bound to protein from free (/sup 3/H)forskolin by rapid filtration. The K/sub d/ for (/sup 3/H)forskolin binding to solubilized proteins was 14 nM which was similar to that for (/sup 3/H)forskolin binding sites in membranes from rat brain and human platelets. Forskolin analogs competed for (/sup 3/H)forskolin binding sites with the same rank potency in both brain membranes and in proteins solubilized from brain membranes. (/sup 3/H)forskolin bound to proteins solubilized from membranes with a Bmaxmore » of 38 fmolmg protein which increased to 94 fmolmg protein when GppNHp was included in the binding assay. In contrast, GppNHp had no effect on (/sup 3/H)forskolin binding to proteins solubilized from membranes preactivated with GppNHp. Solubilized adenylate cyclase from non-preactivated membranes had a basal activity of 130 pmolmgmin which was increased 7-fold by GppNHp. In contrast, adenylate cyclase from preactivated membranes had a basal activity of 850 pmolmgmin which was not stimulated by GppNHp or forskolin« less

Deconstructing the DGAT1 enzyme: membrane interactions at substrate binding sites.

PubMed

Lopes, Jose L S; Beltramini, Leila M; Wallace, Bonnie A; Araujo, Ana P U

2015-01-01

Diacylglycerol acyltransferase 1 (DGAT1) is a key enzyme in the triacylglyceride synthesis pathway. Bovine DGAT1 is an endoplasmic reticulum membrane-bound protein associated with the regulation of fat content in milk and meat. The aim of this study was to evaluate the interaction of DGAT1 peptides corresponding to putative substrate binding sites with different types of model membranes. Whilst these peptides are predicted to be located in an extramembranous loop of the membrane-bound protein, their hydrophobic substrates are membrane-bound molecules. In this study, peptides corresponding to the binding sites of the two substrates involved in the reaction were examined in the presence of model membranes in order to probe potential interactions between them that might influence the subsequent binding of the substrates. Whilst the conformation of one of the peptides changed upon binding several types of micelles regardless of their surface charge, suggesting binding to hydrophobic domains, the other peptide bound strongly to negatively-charged model membranes. This binding was accompanied by a change in conformation, and produced leakage of the liposome-entrapped dye calcein. The different hydrophobic and electrostatic interactions observed suggest the peptides may be involved in the interactions of the enzyme with membrane surfaces, facilitating access of the catalytic histidine to the triacylglycerol substrates.

Lecithin retinol acyltransferase and its S175R mutant have a similar secondary structure content and maximum insertion pressure but different enzyme activities.

PubMed

Bussières, Sylvain; Cantin, Line; Salesse, Christian

2011-11-01

Recent work on Lecithin:retinol acyltransferase (LRAT) allowed to gather a large amount of information on its secondary structure, enzymatic properties and membrane binding. A truncated form of LRAT (tLRAT) as well as its S175R mutant leading to retinis pigmentosa, a severe form of retinal dystrophy, were studied to understand the role of this mutation on the dysfunction of this protein. Consistently with previous reports, the S175R-tLRAT mutant was shown to lack enzyme activity. However, very similar secondary structures probed by circular dichroism have been obtained with the S175R-tLRAT mutant and tLRAT. Moreover, similar values of maximum insertion pressure of the S175R-tLRAT mutant and tLRAT have been obtained using Langmuir monolayers, thus suggesting that the S175R mutation has no effect on the membrane binding properties of tLRAT. These findings leave open the possibility that the loss of enzymatic activity associated with the S175R mutant is related to loss of an essential nucleophile near the active site, or alternatively, to steric obstruction of the active site that impedes substrate binding. Copyright © 2011 Elsevier Ltd. All rights reserved.

Plasma membrane calcium ATPases and related disorders.

PubMed

Giacomello, Marta; De Mario, Agnese; Scarlatti, Chiara; Primerano, Simona; Carafoli, Ernesto

2013-03-01

The plasma membrane Ca(2+) ATPases (PMCA pumps) cooperate with other transport systems in the plasma membrane and in the organelles in the regulation of cell Ca(2+). They have high Ca(2+) affinity and are thus the fine tuners of cytosolic Ca(2+). They belong to the superfamily of P-type ATPases: their four basic isoforms share the essential properties of the reaction cycle and the general membrane topography motif of 10 transmembrane domains and three large cytosolic units. However they also differ in other important properties, e.g., tissue distribution and regulatory mechanisms. Their chief regulator is calmodulin, that removes their C-terminal cytosolic tail from autoinhibitory binding sites next to the active site of the pump, restoring activity. The number of pump isoforms is increased to over 30 by alternative splicing of the transcripts at a N-terminal site (site A) and at site C within the C-terminal calmodulin binding domain: the splice variants are tissue specific and developmentally regulated. The importance of PMCAs in the maintenance of cellular Ca(2+) homeostasis is underlined by the disease phenotypes, genetic or acquired, caused by their malfunction. Non-genetic PMCA deficiencies have long been considered possible causative factors in disease conditions as important as cancer, hypertension, or neurodegeneration. Those of genetic origin are better characterized: some have now been discovered in humans as well. They concern all four PMCA isoforms, and range from cardiac dysfunctions, to deafness, to hypertension, to cerebellar ataxia. Copyright © 2012 Elsevier Ltd. All rights reserved.

Endothelial glycocalyx: permeability barrier and mechanosensor.

PubMed

Curry, F E; Adamson, R H

2012-04-01

Endothelial cells are covered with a polysaccharide rich layer more than 400 nm thick, mechanical properties of which limit access of circulating plasma components to endothelial cell membranes. The barrier properties of this endothelial surface layer are deduced from the rate of tracer penetration into the layer and the mechanics of red and white cell movement through capillary microvessels. This review compares the mechanosensor and permeability properties of an inner layer (100-150 nm, close to the endothelial membrane) characterized as a quasi-periodic structure which accounts for key aspects of transvascular exchange and vascular permeability with those of the whole endothelial surface layers. We conclude that many of the barrier properties of the whole surface layer are not representative of the primary fiber matrix forming the molecular filter determining transvascular exchange. The differences between the properties of the whole layer and the inner glycocalyx structures likely reflect dynamic aspects of the endothelial surface layer including tracer binding to specific components, synthesis and degradation of key components, activation of signaling pathways in the endothelial cells when components of the surface layer are lost or degraded, and the spatial distribution of adhesion proteins in microdomains of the endothelial cell membrane.

C2 Domain of Protein Kinase Cα: Elucidation of the Membrane Docking Surface by Site-Directed Fluorescence and Spin Labeling†

PubMed Central

Kohout, Susy C.; Corbalán-García, Senena; Gómez-Fernández, Juan C.; Falke, Joseph J.

2013-01-01

The C2 domain is a conserved signaling motif that triggers membrane docking in a Ca2+-dependent manner, but the membrane docking surfaces of many C2 domains have not yet been identified. Two extreme models can be proposed for the docking of the protein kinase Cα (PKCα) C2 domain to membranes. In the parallel model, the membrane-docking surface includes the Ca2+ binding loops and an anion binding site on β-strands 3–4, such that the β-strands are oriented parallel to the membrane. In the perpendicular model, the docking surface is localized to the Ca2+ binding loops and the β-strands are oriented perpendicular to the membrane surface. The present study utilizes site-directed fluorescence and spin-labeling to map out the membrane docking surface of the PKCα C2 domain. Single cysteine residues were engineered into 18 locations scattered over all regions of the protein surface, and were used as attachment sites for spectroscopic probes. The environmentally sensitive fluorescein probe identified positions where Ca2+ activation or membrane docking trigger measurable fluorescence changes. Ca2+ binding was found to initiate a global conformational change, while membrane docking triggered the largest fluorescein environmental changes at labeling positions on the three Ca2+ binding loops (CBL), thereby localizing these loops to the membrane docking surface. Complementary EPR power saturation measurements were carried out using a nitroxide spin probe to determine a membrane depth parameter, Φ, for each spin-labeled mutant. Positive membrane depth parameters indicative of membrane insertion were found for three positions, all located on the Ca2+ binding loops: N189 on CBL 1, and both R249 and R252 on CBL 3. In addition, EPR power saturation revealed that five positions near the anion binding site are partially protected from collisions with an aqueous paramagnetic probe, indicating that the anion binding site lies at or near the surface of the headgroup layer. Together, the fluorescence and EPR results indicate that the Ca2+ first and third Ca2+ binding loops insert directly into the lipid headgroup region of the membrane, and that the anion binding site on β-strands 3–4 lies near the headgroups. The data support a model in which the β-strands are tilted toward the parallel orientation relative to the membrane surface. PMID:12564928

Modulation of protein function in membrane mimetics: Characterization of P. denitrificans cNOR in nanodiscs or liposomes.

PubMed

Ter Beek, Josy; Kahle, Maximilian; Ädelroth, Pia

2017-10-01

For detailed functional characterization, membrane proteins are usually studied in detergent. However, it is becoming clear that detergent micelles are often poor mimics of the lipid environment in which these proteins function. In this work we compared the catalytic properties of the membrane-embedded cytochrome c-dependent nitric oxide reductase (cNOR) from Paracoccus (P.) denitrificans in detergent, lipid/protein nanodiscs, and proteoliposomes. We used two different lipid mixtures, an extract of soybean lipids and a defined mix of synthetic lipids mimicking the original P. denitrificans membrane. We show that the catalytic activity of detergent-solubilized cNOR increased threefold upon reconstitution from detergent into proteoliposomes with the P. denitrificans lipid mixture, and above two-fold when soybean lipids were used. In contrast, there was only a small activity increase in nanodiscs. We further show that binding of the gaseous ligands CO and O 2 are affected differently by reconstitution. In proteoliposomes the turnover rates are affected much more than in nanodiscs, but CO-binding is more significantly accelerated in liposomes with soybean lipids, while O 2 -binding is faster with the P. denitrificans lipid mix. We also investigated proton-coupled electron transfer during the reaction between fully reduced cNOR and O 2 , and found that the pK a of the internal proton donor was increased in proteoliposomes but not in nanodiscs. Taking our results together, the liposome-reconstituted enzyme shows significant differences to detergent-solubilized protein. Nanodiscs show much more subtle effects, presumably because of their much lower lipid to protein ratio. Which of these two membrane-mimetic systems best mimics the native membrane is discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

Structural basis of sterol recognition and nonvesicular transport by lipid transfer proteins anchored at membrane contact sites.

PubMed

Tong, Junsen; Manik, Mohammad Kawsar; Im, Young Jun

2018-01-30

Membrane contact sites (MCSs) in eukaryotic cells are hotspots for lipid exchange, which is essential for many biological functions, including regulation of membrane properties and protein trafficking. Lipid transfer proteins anchored at membrane contact sites (LAMs) contain sterol-specific lipid transfer domains [StARkin domain (SD)] and multiple targeting modules to specific membrane organelles. Elucidating the structural mechanisms of targeting and ligand recognition by LAMs is important for understanding the interorganelle communication and exchange at MCSs. Here, we determined the crystal structures of the yeast Lam6 pleckstrin homology (PH)-like domain and the SDs of Lam2 and Lam4 in the apo form and in complex with ergosterol. The Lam6 PH-like domain displays a unique PH domain fold with a conserved N-terminal α-helix. The Lam6 PH-like domain lacks the basic surface for phosphoinositide binding, but contains hydrophobic patches on its surface, which are critical for targeting to endoplasmic reticulum (ER)-mitochondrial contacts. Structures of the LAM SDs display a helix-grip fold with a hydrophobic cavity and a flexible Ω1-loop as a lid. Ergosterol is bound to the pocket in a head-down orientation, with its hydrophobic acyl group located in the tunnel entrance. The Ω1-loop in an open conformation is essential for ergosterol binding by direct hydrophobic interaction. Structural comparison suggested that the sterol binding mode of the Lam2 SD2 is likely conserved among the sterol transfer proteins of the StARkin superfamily. Structural models of full-length Lam2 correlated with the sterol transport function at the membrane contact sites.

Functional reconstitution of the voltage-regulated sodium channel purified from electroplax of Electrophorus electricus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rosenberg, R.L.

1985-01-01

The voltage-regulated NA channel is responsible for the depolarization of the excitable cell membrane during the normal action potential. This research has focused on the functional properties of the Na channel, purified from detergent extracts of electroplax membranes of the electric eel, and reconstituted into vesicles of defined phospholipid. These properties were assessed by measuring neurotoxin-modulated ion flux into the reconstituted membrane vesicles and by recording the single-channel currents of the purified channel by the patch-clamp method. The binding of tritiated tetrodotoxin (TTX) was employed as a marker for the purification of the channel. Two high-resolution fractionation steps, based onmore » molecular charge and protein size, were used to obtain a preparation that is 80% homogeneous for a large peptide of 270,000 daltons. Radiotracer /sup 22/Na/sup +/ influx into the vesicles was stimulated by veratridine and by batrachotoxin (BTX) at concentrations of 100 ..mu..M and 5 ..mu..M, respectively. The stimulation by BTX was greater than that by veratridine, and can be as much as 16-fold over control influx levels. The stimulated influx is blocked by TTX with a K/sub i/ of 35 nM, and by local anesthetics in the normal pharmacological range. Large multilamellar vesicles prepared with a freeze-thaw step are suitable for single-channel recording techniques. When excised patches of the reconstituted membranes were voltage-clamped in the absence of activating neurotoxins, voltage-dependent single-channel currents were recorded. These displayed properties similar to those from native membranes of nerve and muscle. These results indicate that the protein purified on the basis of TTX binding is a functional Na channel possessing these functional domains: the ion-selective channel, the voltage sensors controlling activation and inactivation, and the sites of action of TTX, alkaloid neurotoxins, and local anesthetics.« less

Insights into the complex association of bovine factor Va with acidic-lipid-containing synthetic membranes.

PubMed Central

Cutsforth, G A; Koppaka, V; Krishnaswamy, S; Wu, J R; Mann, K G; Lentz, B R

1996-01-01

The mechanism of binding of blood coagulation cofactor factor Va to acidic-lipid-containing membranes has been addressed. Binding isotherms were generated at room temperature using the change in fluorescence anisotropy of pyrene-labeled bovine factor Va to detect binding to sonicated membrane vesicles containing either bovine brain phosphatidylserine (PS) or 1,2-dioleoyl-3-sn-phosphatidylglycerol (DOPG) in combination with 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC). The composition of the membranes was varied from 0 to 40 mol% for PS/POPC and from 0 to 65 mol % for DOPG/POPC membranes. Fitting the data to a classical Langmuir adsorption model yielded estimates of the dissociation constant (Kd) and the stoichiometry of binding. The values of Kd defined in this way displayed a maximum at low acidic lipid content but were nearly constant at intermediate to high fractions of acidic lipid. Fitting the binding isotherms to a two-process binding model (nonspecific adsorption in addition to binding of acidic lipids to sites on the protein) suggested a significant acidic-lipid-independent binding affinity in addition to occupancy of three protein sites that bind PS in preference to DOPG. Both analyses indicated that interaction of factor Va with an acidic-lipid-containing membrane is much more complex than those of factor Xa or prothrombin. Furthermore, a change in the conformation of bound pyrene-labeled factor Va with surface concentration of acidic lipid was implied by variation of both the saturating fluorescence anisotropy and the binding parameters with the acidic lipid content of the membrane. Finally, the results cannot support the contention that binding occurs through nonspecific adsorption to a patch or domain of acidic lipids in the membrane. Factor Va is suggested to associate with membranes by a complex process that includes both acidic-lipid-specific and acidic-lipid-independent sites and a protein structure change induced by occupancy of acidic-lipid-specific sites on the factor Va molecule. Images FIGURE 5 PMID:8744332

Enhancement in Proton Conductivity and Thermal Stability in Nafion Membranes Induced by Incorporation of Sulfonated Carbon Nanotubes.

PubMed

Yin, Chongshan; Li, Jingjing; Zhou, Yawei; Zhang, Haining; Fang, Pengfei; He, Chunqing

2018-04-25

Proton exchange membrane fuel cell (PEMFC) is one of the most promising green power sources, in which perfluorinated sulfonic acid ionomer-based membranes (e.g., Nafion) are widely used. However, the widespread application of PEMFCs is greatly limited by the sharp degradation in electrochemical properties of the proton exchange membranes under high temperature and low humidity conditions. In this work, the high-performance sulfonated carbon nanotubes/Nafion composite membranes (Su-CNTs/Nafion) for the PEMFCs were prepared and the mechanism of the microstructures on the macroscopic properties of membranes was intensively studied. Microstructure evolution in Nafion membranes during water uptake was investigated by positron annihilation lifetime spectroscopy, and results strongly showed that the Su-CNTs or CNTs in Nafion composite membranes significantly reinforced Nafion matrices, which influenced the development of ionic-water clusters in them. Proton conductivities in Su-CNTs/Nafion composite membranes were remarkably enhanced due to the mass formation of proton-conducting pathways (water channels) along the Su-CNTs. In particular, these pathways along Su-CNTs in Su-CNTs/Nafion membranes interconnected the isolated ionic-water clusters at low humidity and resulted in less tortuosity of the water channel network for proton transportation at high humidity. At a high temperature of 135 °C, Su-CNTs/Nafion membranes maintained high proton conductivity because the reinforcement of Su-CNTs on Nafion matrices reduced the evaporation of water molecules from membranes as well as the hydrophilic Su-CNTs were helpful for binding water molecules.

Photoaffinity labelling and solubilization on the central 5-HT1A receptor binding site.

PubMed

Gozlan, H; Emerit, M B; el Mestikawy, S; Cossery, J M; Marquet, A; Besselievre, R; Hamon, M

1987-01-01

Two complementary approaches, covalent labelling and solubilization, have been used to study the biochemical properties of the central 5-HT1A receptor binding site. We have first designed a photoaffinity ligand containing the structure of 8-OH-DPAT, a potent and specific agonist of 5-HT1A sites. Thus, 8-methoxy-2[N-n-propyl,N-3-(2-nitro-4-azido-phenyl)- aminopropyl]aminotetralin or 8-methoxy-3'-NAP-amino-PAT, was found to displace, in the dark, [3H]8-OH-DPAT from 5-HT1A sites in rat hippocampal membranes with an IC50 of 6.6 nM. Under two cumulative UV irradiations (366 nm, for 20 min at 4 degrees C), 8-methoxy-3-'-NAP-amino-PAT (30 nM) blocked irreversibly 55-60% of 5-HT1A binding sites. This blockade was specific of 5-HT1A sites since the other serotoninergic sites, 5-HT1B, 5-HT2 and also the presynaptic 5-HT3 sites were not affected by the treatment. In addition, the binding of [3H]Spiperone and [3H]7-OH-DPAT to striatal dopamine sites remained unchanged under similar photolysis conditions. The tritiated derivative of the photoaffinity ligand (92 Ci/mmol) was then synthesized for the identification of the covalently bound protein(s). SDS-PAGE of solubilized membranes irradiated in the presence of 20 nM 3H-8-methoxy-3'-NAP-amino-PAT allowed the detection of a 63 kD protein whose labelling appeared specific. Thus, 3H-incorporation into the 63 kD band could be prevented by microM concentrations of 5-HT, 8-OH-DPAT and other selective 5-HT1A ligands such as isapirone. In contrast, the 5-HT2 antagonist ketanserin, norepinephrine and dopamine-related ligands (including 7-OH-DPAT) were ineffective. Direct solubilization of 5-HT1A receptor binding sites was also attempted from rat hippocampal membranes. The best results were obtained using CHAPS (10 mM) plus NaCl (0.2 M), which led to 50% recovery of 5-HT1A sites in the 100,000 g supernatant. The pharmacological properties and sensitivity to N-ethyl-maleimide and GppNHp of soluble sites appeared near identical to those of membrane-bound 5-HT1A sites.

Cyclotides Insert into Lipid Bilayers to Form Membrane Pores and Destabilize the Membrane through Hydrophobic and Phosphoethanolamine-specific Interactions*

PubMed Central

Wang, Conan K.; Wacklin, Hanna P.; Craik, David J.

2012-01-01

Cyclotides are a family of plant-derived circular proteins with potential therapeutic applications arising from their remarkable stability, broad sequence diversity, and range of bioactivities. Their membrane-binding activity is believed to be a critical component of their mechanism of action. Using isothermal titration calorimetry, we studied the binding of the prototypical cyclotides kalata B1 and kalata B2 (and various mutants) to dodecylphosphocholine micelles and phosphoethanolamine-containing lipid bilayers. Although binding is predominantly an entropy-driven process, suggesting that hydrophobic forces contribute significantly to cyclotide-lipid complex formation, specific binding to the phosphoethanolamine-lipid headgroup is also required, which is evident from the enthalpic changes in the free energy of binding. In addition, using a combination of dissipative quartz crystal microbalance measurements and neutron reflectometry, we elucidated the process by which cyclotides interact with bilayer membranes. Initially, a small number of cyclotides bind to the membrane surface and then insert first into the outer membrane leaflet followed by penetration through the membrane and pore formation. At higher concentrations of cyclotides, destabilization of membranes occurs. Our results provide significant mechanistic insight into how cyclotides exert their bioactivities. PMID:23129773

Analysis of Perforin Assembly by Quartz Crystal Microbalance Reveals a Role for Cholesterol and Calcium-independent Membrane Binding.

PubMed

Stewart, Sarah E; Bird, Catherina H; Tabor, Rico F; D'Angelo, Michael E; Piantavigna, Stefania; Whisstock, James C; Trapani, Joseph A; Martin, Lisandra L; Bird, Phillip I

2015-12-25

Perforin is an essential component in the cytotoxic lymphocyte-mediated cell death pathway. The traditional view holds that perforin monomers assemble into pores in the target cell membrane via a calcium-dependent process and facilitate translocation of cytotoxic proteases into the cytoplasm to induce apoptosis. Although many studies have examined the structure and role of perforin, the mechanics of pore assembly and granzyme delivery remain unclear. Here we have employed quartz crystal microbalance with dissipation monitoring (QCM-D) to investigate binding and assembly of perforin on lipid membranes, and show that perforin monomers bind to the membrane in a cooperative manner. We also found that cholesterol influences perforin binding and activity on intact cells and model membranes. Finally, contrary to current thinking, perforin efficiently binds membranes in the absence of calcium. When calcium is added to perforin already on the membrane, the QCM-D response changes significantly, indicating that perforin becomes membranolytic only after calcium binding. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Effect of resveratrol or ascorbic acid on the stability of α-tocopherol in O/W emulsions stabilized by whey protein isolate: Simultaneous encapsulation of the vitamin and the protective antioxidant.

PubMed

Wang, Lei; Gao, Yahui; Li, Juan; Subirade, Muriel; Song, Yuanda; Liang, Li

2016-04-01

Food proteins have been widely used as carrier materials due to their multiple functional properties. Hydrophobic bioactives are generally dissolved in the oil phase of O/W emulsions. Ligand-binding properties provide the possibility of binding bioactives to the protein membrane of oil droplets. In this study, the influence of whey protein isolate (WPI) concentration and amphiphilic resveratrol or hydrophilic ascorbic acid on the decomposition of α-tocopherol in the oil phase of WPI emulsions is considered. Impact of ascorbic acid, in the continuous phase, on the decomposition depended on the vitamin concentration. Resveratrol partitioned into the oil-water interface and the cis-isomer contributed most of the protective effect of this polyphenol. About 94% of α-tocopherol and 50% of resveratrol were found in the oil droplets stabilized by 0.01% WPI. These results suggest the feasibility of using the emulsifying and ligand-binding properties of WPI to produce carriers for simultaneous encapsulation of bioactives with different physicochemical properties. Copyright © 2015 Elsevier Ltd. All rights reserved.

Functional dynamics of cell surface membrane proteins

NASA Astrophysics Data System (ADS)

Nishida, Noritaka; Osawa, Masanori; Takeuchi, Koh; Imai, Shunsuke; Stampoulis, Pavlos; Kofuku, Yutaka; Ueda, Takumi; Shimada, Ichio

2014-04-01

Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules.

Functional dynamics of cell surface membrane proteins.

PubMed

Nishida, Noritaka; Osawa, Masanori; Takeuchi, Koh; Imai, Shunsuke; Stampoulis, Pavlos; Kofuku, Yutaka; Ueda, Takumi; Shimada, Ichio

2014-04-01

Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules. Copyright © 2013 Elsevier Inc. All rights reserved.

Molecular modeling of biomembranes and their complexes with protein transmembrane α-helices

NASA Astrophysics Data System (ADS)

Kuznetsov, Andrey S.; Smirnov, Kirill V.; Antonov, Mikhail Yu.; Nikolaev, Ivan N.; Efremov, Roman G.

2017-11-01

Helical segments are common structural elements of membrane proteins. Dimerization and oligomerization of transmembrane (TM) α-helices provides the framework for spatial structure formation and protein-protein interactions. The membrane itself also takes part in the protein functioning. There are some examples of the mutual influence of the lipid bilayer properties and embedded membrane proteins. This work aims at the detail investigation of protein-lipid interactions using model systems: TM peptides corresponding to native protein segments. Three peptides were considered corresponding to TM domains of human glycophorin A (GpA), epidermal growth factor receptor (EGFR) and proposed TM-segment of human neuraminidase-1 (Neu1). A computational analysis of structural and dynamical properties was performed using molecular dynamics method. Monomers of peptides were considered incorporated into hydrated lipid bilayers. It was confirmed, that all these TM peptides have stable helical conformation in lipid environment, and the mutual adaptation of peptides and membrane was observed. It was shown that incorporation of the peptide into membrane results in the modulation of local and mean structural properties of the bilayer. Each peptide interacts with lipid acyl chains having special binding sites on the surface of central part of α-helix that exist for at least 200 ns. However, lipid acyl chains substitute each other faster occupying the same site. The formation of a special pattern of protein-lipid interactions may modulate the association of TM domains of membrane proteins, so membrane environment should be considered when proposing new substances targeting cell receptors.

Molecular dynamics simulations of salicylate effects on the micro- and mesoscopic properties of a dipalmitoylphosphatidylcholine bilayer†

PubMed Central

Song, Yuhua; Guallar, Victor; Baker, Nathan A.

2008-01-01

Salicylate, an amphiphilic molecule and a popular member of non-steroidal antiinflammatory drug family, is known to affect hearing through reduction of the electromechanical coupling in the outer hair cells of the ear. This reduction of electromotility by salicylate has been widely studied but the molecular mechanism of the phenomenon is still unknown. In this study, we investigated one aspect of salicylate’s action; namely, the perturbation of electrical and mechanical membrane properties by salicylate in the absence of cytoskeletal or membrane-bound motor proteins such as prestin. In particular, we simulated the interaction of salicylate with a dipalmitoylphosphatidylcholine (DPPC) bilayer via atomically-detailed molecular dynamics simulations to observe the effect of salicylate on the microscopic and mesoscopic properties of the bilayer. The results demonstrate that salicylate interacts with the bilayer by associating at the water-DPPC interface in a nearly perpendicular orientation and penetrating more deeply into the bilayer than either sodium or chloride. This association has several affects on the membrane properties. First, binding of salicylate to the membrane displaces chloride from the bilayer-water interface. Second, salicylate influences the electrostatic potential and dielectric properties of the bilayer, with significant changes at the water-lipid bilayer interface. Third, salicylate association results in structural changes including decreased head group area per lipid and increased lipid tail order. However, salicylate does not significantly alter the mechanical properties of the DPPC bilayer; bulk compressibility, area compressibility, and bending modulus were only perturbed by small, statistically-insignificant amounts, by the presence of salicylate. The observations from these simulations are in qualitative agreement with experimental data and support the conclusion that salicylate influences the electrical but not the mechanical properties of DPPC membranes. PMID:16216066

[Molecular organization of glutamate-sensitive chemoexcitatory membranes of nerve cells. Binding of L-[3H]glutamate to synaptic membranes of the rat cerebral cortex].

PubMed

Dambinova, S A; Gorodinskiĭ, A I

1984-01-01

The binding of L-[3H]glutamate to rat cerebral cortex synaptic membranes was investigated. Two types of binding sites, a Na+-independent (Kd = 140-160 nm; Bmax = 3.8-4.5 pmol-mg of protein) and a Na+-dependent (Kd = 2.0 microM; Bmax = 45-50 pmol/mg of protein) ones, were detected. The dependence of Na+-insensitive binding on time and temperature and membrane content in a sample was determined. Mono- and divalent cations (5-10 mM) potentiated specific binding by 2.1-3.3 times. The Na+-dependent binding is associated with active transport systems, while the Na+-independent one-with true receptor binding. The relationship between CNS glutamate receptors and Na+-independent binding sites is discussed.

The F-BAR domains from srGAP1, srGAP2 and srGAP3 regulate membrane deformation differently

PubMed Central

Coutinho-Budd, Jaeda; Ghukasyan, Vladimir; Zylka, Mark J.; Polleux, Franck

2012-01-01

Summary Coordination of membrane deformation and cytoskeletal dynamics lies at the heart of many biological processes critical for cell polarity, motility and morphogenesis. We have recently shown that Slit-Robo GTPase-activating protein 2 (srGAP2) regulates neuronal morphogenesis through the ability of its F-BAR domain to regulate membrane deformation and induce filopodia formation. Here, we demonstrate that the F-BAR domains of two closely related family members, srGAP1 and srGAP3 [designated F-BAR(1) and F-BAR(3), respectively] display significantly different membrane deformation properties in non-neuronal COS7 cells and in cortical neurons. F-BAR(3) induces filopodia in both cell types, though less potently than F-BAR(2), whereas F-BAR(1) prevents filopodia formation in cortical neurons and reduces plasma membrane dynamics. These three F-BAR domains can heterodimerize, and they act synergistically towards filopodia induction in COS7 cells. As measured by fluorescence recovery after photobleaching, F-BAR(2) displays faster molecular dynamics than F-BAR(3) and F-BAR(1) at the plasma membrane, which correlates well with its increased potency to induce filopodia. We also show that the molecular dynamic properties of F-BAR(2) at the membrane are partially dependent on F-Actin. Interestingly, acute phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] depletion in cells does not interfere with plasma membrane localization of F-BAR(2), which is compatible with our result showing that F-BAR(2) binds to a broad range of negatively-charged phospholipids present at the plasma membrane, including phosphatidylserine (PtdSer). Overall, our results provide novel insights into the functional diversity of the membrane deformation properties of this subclass of F-BAR-domains required for cell morphogenesis. PMID:22467852

Interaction of Antiinflammatory Drugs with EPC Liposomes: Calorimetric Study in a Broad Concentration Range

PubMed Central

Matos, Carla; Lima, José L. C.; Reis, Salette; Lopes, António; Bastos, Margarida

2004-01-01

Isothermal titration calorimetry was used to characterize and quantify the partition of indomethacin and acemetacin between the bulk aqueous phase and the membrane of egg phosphatidylcholine vesicles. Significant electrostatic effects were observed due to binding of the charged drugs to the membrane, which implied the use of the Gouy-Chapman theory to calculate the interfacial concentrations. The binding/partition phenomenon was quantified in terms of the partition coefficient (Kp), and/or the equilibrium constant (Kb). Mathematical expressions were developed, either to encompass the electrostatic effects in the partition model, or to numerically relate partition coefficients and binding constants. Calorimetric titrations conducted under a lipid/drug ratio >100:1 lead to a constant heat release and were used to directly calculate the enthalpy of the process, ΔH, and indirectly, ΔG and ΔS. As the lipid/drug ratio decreased, the constancy of reaction enthalpy was tested in the fitting process. Under low lipid/drug ratio conditions simple partition was no longer valid and the interaction phenomenon was interpreted in terms of binding isotherms. A mathematical expression was deduced for quantification of the binding constants and the number of lipid molecules associated with one drug molecule. The broad range of concentrations used stressed the biphasic nature of the interaction under study. As the lipid/drug ratio was varied, the results showed that the interaction of both drugs does not present a unique behavior in all studied regimes: the extent of the interaction, as well as the binding stoichiometry, is affected by the lipid/drug ratio. The change in these parameters reflects the biphasic behavior of the interaction—possibly the consequence of a modification of the membrane's physical properties as it becomes saturated with the drug. PMID:14747330

Two interdependent mechanisms of antimicrobial activity allow for efficient killing in nylon-3-based polymeric mimics of innate immunity peptides.

PubMed

Lee, Michelle W; Chakraborty, Saswata; Schmidt, Nathan W; Murgai, Rajan; Gellman, Samuel H; Wong, Gerard C L

2014-09-01

Novel synthetic mimics of antimicrobial peptides have been developed to exhibit structural properties and antimicrobial activity similar to those of natural antimicrobial peptides (AMPs) of the innate immune system. These molecules have a number of potential advantages over conventional antibiotics, including reduced bacterial resistance, cost-effective preparation, and customizable designs. In this study, we investigate a family of nylon-3 polymer-based antimicrobials. By combining vesicle dye leakage, bacterial permeation, and bactericidal assays with small-angle X-ray scattering (SAXS), we find that these polymers are capable of two interdependent mechanisms of action: permeation of bacterial membranes and binding to intracellular targets such as DNA, with the latter necessarily dependent on the former. We systemically examine polymer-induced membrane deformation modes across a range of lipid compositions that mimic both bacteria and mammalian cell membranes. The results show that the polymers' ability to generate negative Gaussian curvature (NGC), a topological requirement for membrane permeation and cellular entry, in model Escherichia coli membranes correlates with their ability to permeate membranes without complete membrane disruption and kill E. coli cells. Our findings suggest that these polymers operate with a concentration-dependent mechanism of action: at low concentrations permeation and DNA binding occur without membrane disruption, while at high concentrations complete disruption of the membrane occurs. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova. Copyright © 2014 Elsevier B.V. All rights reserved.

Interactions between peroxiredoxin 2, hemichrome and the erythrocyte membrane.

PubMed

Bayer, Simone B; Low, Felicia M; Hampton, Mark B; Winterbourn, Christine C

2016-12-01

Peroxiredoxin 2 (Prx2) is an abundant antioxidant protein in erythrocytes that protects against hemolytic anemia resulting from hemoglobin oxidation and Heinz body formation. A small fraction of Prx2 is bound to the cell membrane, but the mechanism and relevance of binding are not clear. We have investigated Prx2 interactions with the erythrocyte membrane and oxidized hemoglobin and whether these interactions are dependent on Prx2 redox state. Membrane binding of Prx2 in erythrocytes decreased when the cells were treated with H 2 O 2 , but studies with purified Prx2 and isolated ghosts showed that the interaction was independent of Prx2 redox state. Hemoglobin oxidation leads to the formation of hemichrome, a denatured form of the protein that binds to Band3 protein in the cell membrane as part of the senescence process and is a precursor of Heinz bodies. Hemichrome competed with Prx2 and decreased Prx2 binding to the membrane, potentially explaining the decreased binding in oxidant-exposed cells. The increased membrane binding of Prx2 seen with increasing intracellular calcium was less sensitive to H 2 O 2 or hemichrome, suggesting an alternative mode of binding. Prx2 was also shown to exhibit chaperone-like activity by retarding the precipitation of pre-formed hemichrome. Our results suggest that Prx2, by restricting membrane binding of hemichrome, could impede Band3 clustering and exposure of senescence antigens. This mechanism, plus the observed chaperone activity for oxidized hemoglobin, may help protect against hemolytic anemia.

Determining Membrane Protein-Lipid Binding Thermodynamics Using Native Mass Spectrometry.

PubMed

Cong, Xiao; Liu, Yang; Liu, Wen; Liang, Xiaowen; Russell, David H; Laganowsky, Arthur

2016-04-06

Membrane proteins are embedded in the biological membrane where the chemically diverse lipid environment can modulate their structure and function. However, the thermodynamics governing the molecular recognition and interaction of lipids with membrane proteins is poorly understood. Here, we report a method using native mass spectrometry (MS), to determine thermodynamics of individual ligand binding events to proteins. Unlike conventional methods, native MS can resolve individual ligand binding events and, coupled with an apparatus to control the temperature, determine binding thermodynamic parameters, such as for protein-lipid interactions. We validated our approach using three soluble protein-ligand systems (maltose binding protein, lysozyme, and nitrogen regulatory protein) and obtained similar results to those using isothermal titration calorimetry and surface plasmon resonance. We also determined for the first time the thermodynamics of individual lipid binding to the ammonia channel (AmtB), an integral membrane protein from Escherichia coli. Remarkably, we observed distinct thermodynamic signatures for the binding of different lipids and entropy-enthalpy compensation for binding lipids of variable chain length. Additionally, using a mutant form of AmtB that abolishes a specific phosphatidylglycerol (PG) binding site, we observed distinct changes in the thermodynamic signatures for binding PG, implying these signatures can identify key residues involved in specific lipid binding and potentially differentiate between specific lipid binding sites.

Mechanism of antifungal activity of antimicrobial peptide APP, a cell-penetrating peptide derivative, against Candida albicans: intracellular DNA binding and cell cycle arrest.

PubMed

Li, Lirong; Sun, Jin; Xia, Shufang; Tian, Xu; Cheserek, Maureen Jepkorir; Le, Guowei

2016-04-01

We investigated the antifungal properties and anti-candidal mechanism of antimicrobial peptide APP. The minimum inhibitory concentration of APP was 8 μM against Candida albicans and Aspeogillus flavus, the concentration against Saccharomyces cerevisiae and Cryptococcus neoformans was 16 μM, while 32 μM inhibited Aspergilla niger and Trichopyton rubrum. APP caused slight depolarization (12.32 ± 0.87%) of the membrane potential of intact C. albicans cells when it exerted its anti-candidal activity and only caused 21.52 ± 0.48% C. albicans cell membrane damage. APP interacted with cell wall membrane, caused potassium efflux and nucleotide leakage. However, confocal fluorescence microscopy experiment and flow cytometry confirmed that FITC-labeled APP penetrated C. albicans cell membrane with 52.31 ± 1.88% cell-penetrating efficiency and accumulated in the cytoplasm. Then, APP interact with C. albicans genomic DNA and completely suppressed DNA migration above weight ratio (peptide/DNA) of 2, and significantly arrested cell cycles during the S-phase (S-phase cell population was 27.09 ± 0.73%, p < 0.05) after penetrating the cell membrane. Results indicated that APP kills C. albicans for efficient cell-penetrating efficiency, strong DNA-binding affinity and significant physiological changes inducing S-phase arrest in intracellular environment.

Interaction of sodium dodecyl sulfate with watermelon chromoplasts and examination of the organization of lycopene within the chromoplasts.

PubMed

Fish, Wayne W

2006-10-18

The properties of plant-derived precipitates of watermelon lycopene were examined in aqueous sodium dodecyl sulfate (SDS) as part of an ongoing effort to develop simpler, more economical ways to quantify carotenoids in melon fruit. Levels of SDS >0.2% were found to increase the water solubility of lycopene in the state in which it was isolated from watermelon. Electron microscopy and chemical analyses suggested that the watermelon lycopene as isolated is packaged inside a membrane to form a chromoplast. Spectral peaks in the visible region of the watermelon chromoplasts in SDS exhibited a bathochromic shift from those in organic solvent. Watermelon chromoplasts in SDS exhibited pronounced circular dichroic activity in the visible region. Binding measurements indicated that about 120 molecules of SDS were bound per molecule of lycopene inside the chromoplast; likely, the detergent molecules are bound to the chromoplast membrane. Around 80% of the chromoplast-SDS complexes were retained on a 0.45 mum membrane filter. Together, these observations are consistent with lycopene in a J-type chiral arrangement inside a membrane to form a chromoplast. The binding of SDS molecules to the chromoplast membrane form a complex that is extensively more water-soluble than the chromoplast alone.

Phytochemicals perturb membranes and promiscuously alter protein function.

PubMed

Ingólfsson, Helgi I; Thakur, Pratima; Herold, Karl F; Hobart, E Ashley; Ramsey, Nicole B; Periole, Xavier; de Jong, Djurre H; Zwama, Martijn; Yilmaz, Duygu; Hall, Katherine; Maretzky, Thorsten; Hemmings, Hugh C; Blobel, Carl; Marrink, Siewert J; Koçer, Armağan; Sack, Jon T; Andersen, Olaf S

2014-08-15

A wide variety of phytochemicals are consumed for their perceived health benefits. Many of these phytochemicals have been found to alter numerous cell functions, but the mechanisms underlying their biological activity tend to be poorly understood. Phenolic phytochemicals are particularly promiscuous modifiers of membrane protein function, suggesting that some of their actions may be due to a common, membrane bilayer-mediated mechanism. To test whether bilayer perturbation may underlie this diversity of actions, we examined five bioactive phenols reported to have medicinal value: capsaicin from chili peppers, curcumin from turmeric, EGCG from green tea, genistein from soybeans, and resveratrol from grapes. We find that each of these widely consumed phytochemicals alters lipid bilayer properties and the function of diverse membrane proteins. Molecular dynamics simulations show that these phytochemicals modify bilayer properties by localizing to the bilayer/solution interface. Bilayer-modifying propensity was verified using a gramicidin-based assay, and indiscriminate modulation of membrane protein function was demonstrated using four proteins: membrane-anchored metalloproteases, mechanosensitive ion channels, and voltage-dependent potassium and sodium channels. Each protein exhibited similar responses to multiple phytochemicals, consistent with a common, bilayer-mediated mechanism. Our results suggest that many effects of amphiphilic phytochemicals are due to cell membrane perturbations, rather than specific protein binding.

Phytochemicals Perturb Membranes and Promiscuously Alter Protein Function

PubMed Central

2015-01-01

A wide variety of phytochemicals are consumed for their perceived health benefits. Many of these phytochemicals have been found to alter numerous cell functions, but the mechanisms underlying their biological activity tend to be poorly understood. Phenolic phytochemicals are particularly promiscuous modifiers of membrane protein function, suggesting that some of their actions may be due to a common, membrane bilayer-mediated mechanism. To test whether bilayer perturbation may underlie this diversity of actions, we examined five bioactive phenols reported to have medicinal value: capsaicin from chili peppers, curcumin from turmeric, EGCG from green tea, genistein from soybeans, and resveratrol from grapes. We find that each of these widely consumed phytochemicals alters lipid bilayer properties and the function of diverse membrane proteins. Molecular dynamics simulations show that these phytochemicals modify bilayer properties by localizing to the bilayer/solution interface. Bilayer-modifying propensity was verified using a gramicidin-based assay, and indiscriminate modulation of membrane protein function was demonstrated using four proteins: membrane-anchored metalloproteases, mechanosensitive ion channels, and voltage-dependent potassium and sodium channels. Each protein exhibited similar responses to multiple phytochemicals, consistent with a common, bilayer-mediated mechanism. Our results suggest that many effects of amphiphilic phytochemicals are due to cell membrane perturbations, rather than specific protein binding. PMID:24901212

Biological Activity of Blackcurrant Extracts (Ribes nigrum L.) in Relation to Erythrocyte Membranes

PubMed Central

Cyboran, Sylwia; Żyłka, Romuald; Oszmiański, Jan; Kleszczyńska, Halina

2014-01-01

Compounds contained in fruits and leaves of blackcurrant (Ribes nigrum L.) are known as agents acting preventively and therapeutically on the organism. The HPLC analysis showed they are rich in polyphenol anthocyanins in fruits and flavonoids in leaves, that have antioxidant activity and are beneficial for health. The aim of the research was to determine the effect of blackcurrant fruit and leaf extracts on the physical properties of the erythrocyte membranes and assess their antioxidant properties. The effect of the extracts on osmotic resistance, shape of erythrocytes and hemolytic and antioxidant activity of the extracts were examined with spectrophotometric methods. The FTIR investigation showed that extracts modify the erythrocyte membrane and protect it against free radicals induced by UV radiation. The results show that the extracts do not induce hemolysis and even protect erythrocytes against the harmful action of UVC radiation, while slightly strengthening the membrane and inducing echinocytes. The compounds contained in the extracts do not penetrate into the hydrophobic region, but bind to the membrane surface inducing small changes in the packing arrangement of the polar head groups of membrane lipids. The extracts have a high antioxidant activity. Their presence on the surface of the erythrocyte membrane entails protection against free radicals. PMID:24527456

Biguanides inhibit complex I, II and IV of rat liver mitochondria and modify their functional properties.

PubMed

Drahota, Z; Palenickova, E; Endlicher, R; Milerova, M; Brejchova, J; Vosahlikova, M; Svoboda, P; Kazdova, L; Kalous, M; Cervinkova, Z; Cahova, M

2014-01-01

In this study, we focused on an analysis of biguanides effects on mitochondrial enzyme activities, mitochondrial membrane potential and membrane permeability transition pore function. We used phenformin, which is more efficient than metformin, and evaluated its effect on rat liver mitochondria and isolated hepatocytes. In contrast to previously published data, we found that phenformin, after a 5 min pre-incubation, dose-dependently inhibits not only mitochondrial complex I but also complex II and IV activity in isolated mitochondria. The enzymes complexes inhibition is paralleled by the decreased respiratory control index and mitochondrial membrane potential. Direct measurements of mitochondrial swelling revealed that phenformin increases the resistance of the permeability transition pore to Ca(2+) ions. Our data might be in agreement with the hypothesis of Schäfer (1976) that binding of biguanides to membrane phospholipids alters membrane properties in a non-specific manner and, subsequently, different enzyme activities are modified via lipid phase. However, our measurements of anisotropy of fluorescence of hydrophobic membrane probe diphenylhexatriene have not shown a measurable effect of membrane fluidity with the 1 mM concentration of phenformin that strongly inhibited complex I activity. Our data therefore suggest that biguanides could be considered as agents with high efficacy but low specifity.

Decreased accessibility and lack of activation of ErbB2 in JIMT-1, a herceptin-resistant, MUC4-expressing breast cancer cell line.

PubMed

Nagy, Peter; Friedländer, Elza; Tanner, Minna; Kapanen, Anita I; Carraway, Kermit L; Isola, Jorma; Jovin, Thomas M

2005-01-15

Overexpression of erbB2 in breast tumors is associated with poor prognosis and is a target of receptor-oriented cancer therapy. Trastuzumab (Herceptin), a monoclonal antibody against a membrane-proximal epitope in the extracellular region of erbB2, shows a therapeutic effect against a fraction of erbB2-amplified breast tumors. Unfortunately, resistance to Herceptin is common, and its cause is as yet unclear. Here we investigated the properties of erbB2 in a Herceptin-resistant cell line, JIMT-1, established from a breast cancer patient showing erbB2 gene amplification and primary resistance to Herceptin. The expression profile of erbB proteins, Herceptin-induced erbB2 internalization, and down-regulation in JIMT-1 were similar to those in Herceptin-sensitive lines. However, the mean number of Herceptin Mab binding sites in JIMT-1 was 1/5 that of the expressed erbB2 molecules, although 5% to 10% of the cells showed a approximately 10-fold higher Herceptin binding than the main population. Herceptin Fab and Mab 2C4, an antibody binding to an epitope in the ectodomain further removed from the membrane, bound more efficiently to JIMT-1 cells than Herceptin Mab, implying that erbB2 was partly masked. The expression of MUC4, a membrane-associated mucin that according to reports contributes to the masking of membrane proteins, was higher in JIMT-1 than in Herceptin-sensitive lines, and its level was inversely correlated with the Herceptin binding capacity of single cells. Knockdown of MUC4 expression by RNA interference increased the binding of Herceptin. Western blotting showed a low level of proteolytic processing, shedding, and tyrosine phosphorylation of erbB2 in JIMT-1. The latter finding may explain its Herceptin-resistant phenotype characterizing both the low and high Herceptin binding subpopulations. We conclude that masking of erbB2 in JIMT-1 leads to diminished Herceptin binding and isolation of erbB2 from its normal interaction and activation partners.

APBSmem: A Graphical Interface for Electrostatic Calculations at the Membrane

PubMed Central

Callenberg, Keith M.; Choudhary, Om P.; de Forest, Gabriel L.; Gohara, David W.; Baker, Nathan A.; Grabe, Michael

2010-01-01

Electrostatic forces are one of the primary determinants of molecular interactions. They help guide the folding of proteins, increase the binding of one protein to another and facilitate protein-DNA and protein-ligand binding. A popular method for computing the electrostatic properties of biological systems is to numerically solve the Poisson-Boltzmann (PB) equation, and there are several easy-to-use software packages available that solve the PB equation for soluble proteins. Here we present a freely available program, called APBSmem, for carrying out these calculations in the presence of a membrane. The Adaptive Poisson-Boltzmann Solver (APBS) is used as a back-end for solving the PB equation, and a Java-based graphical user interface (GUI) coordinates a set of routines that introduce the influence of the membrane, determine its placement relative to the protein, and set the membrane potential. The software Jmol is embedded in the GUI to visualize the protein inserted in the membrane before the calculation and the electrostatic potential after completing the computation. We expect that the ease with which the GUI allows one to carry out these calculations will make this software a useful resource for experimenters and computational researchers alike. Three examples of membrane protein electrostatic calculations are carried out to illustrate how to use APBSmem and to highlight the different quantities of interest that can be calculated. PMID:20949122

APBSmem: a graphical interface for electrostatic calculations at the membrane.

PubMed

Callenberg, Keith M; Choudhary, Om P; de Forest, Gabriel L; Gohara, David W; Baker, Nathan A; Grabe, Michael

2010-09-29

Electrostatic forces are one of the primary determinants of molecular interactions. They help guide the folding of proteins, increase the binding of one protein to another and facilitate protein-DNA and protein-ligand binding. A popular method for computing the electrostatic properties of biological systems is to numerically solve the Poisson-Boltzmann (PB) equation, and there are several easy-to-use software packages available that solve the PB equation for soluble proteins. Here we present a freely available program, called APBSmem, for carrying out these calculations in the presence of a membrane. The Adaptive Poisson-Boltzmann Solver (APBS) is used as a back-end for solving the PB equation, and a Java-based graphical user interface (GUI) coordinates a set of routines that introduce the influence of the membrane, determine its placement relative to the protein, and set the membrane potential. The software Jmol is embedded in the GUI to visualize the protein inserted in the membrane before the calculation and the electrostatic potential after completing the computation. We expect that the ease with which the GUI allows one to carry out these calculations will make this software a useful resource for experimenters and computational researchers alike. Three examples of membrane protein electrostatic calculations are carried out to illustrate how to use APBSmem and to highlight the different quantities of interest that can be calculated.

( sup 3 H)RO15-4513 binding to cerebellar diazepam-sensitive and insensitive GABAA receptors is unchanged by one week of ethanol intake

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martin, M.W.; Chen, J.P.; Wallis, C.

1992-02-26

({sup 3}H)RO15-4513, a partial inverse agonist at GABAA receptors, binds to two sites in cerebellar membranes, one sensitive (DZ-S) and one insensitive (DZ-IS) to inhibition by diazepam. These binding sites may represent different isoforms of the GABAA receptor and may play a role in ethanol (EtOH) dependence. The authors tested the hypothesis that chronic intake of EtOH induces changes in the binding properties of one or both of these putative GABBA receptors. Rats were fed a liquid diet of 4.5% EtOH for 7 d, gavaged with a 3g/kg dose of EtOH, and then sacrificed after 2 h, 12 h, ormore » 4.5 d. Binding of ({sup 3}H)RO15-4513 to cerebellar membranes was performed in the absence or presence of 10{mu}M diazepam (DZ-IS binding); DZ-S binding was calculated as the difference between total and DZ-IS. Nonlinear regression analysis showed that each class of binding site fit a model of mass action binding to a single, noninteractive population of sites. No significant difference was observed between any of the treatment groups in the apparent affinity (Kd) for ({sup 3}H)RO15-4513 at total, DZ-S, or DZ-IS sites following chronic EtOH intake or withdrawal. In addition, no significant difference was observed in the apparent number of DZ-S or DZ-IS binding sites or the ratio of DZ-S to DZ-IS.« less

Differential inhibition of [3H]-oxotremorine-M and [3H]-quinuclinidyl benzilate binding to muscarinic receptors in rat brain membranes with acetylcholinesterase inhibitors.

PubMed

Lockhart, B; Closier, M; Howard, K; Steward, C; Lestage, P

2001-04-01

The potential interaction of acetylcholinesterase inhibitors with cholinergic receptors may play a significant role in the therapeutic and/or side-effects associated with this class of compound. In the present study, the capacity of acetylcholinesterase inhibitors to interact with muscarinic receptors was assessed by their ability to displace both [3H]-oxotremorine-M and [3H]-quinuclinidyl benzilate binding in rat brain membranes. The [3H]-quinuclinidyl benzilate/[3H]-oxotremorine-M affinity ratios permitted predictions to be made of either the antagonist or agonist properties of the different compounds. A series of compounds, representative of the principal classes of acetylcholinesterase inhibitors, displaced [3H]-oxotremorine-M binding with high-to-moderate potency (ambenonium>neostigmine=pyridostigmine=tacrine>physostigmine> edrophonium=galanthamine>desoxypeganine) whereas only ambenonium and tacrine displaced [3H]-quinuclinidyl benzilate binding. Inhibitors such as desoxypeganine, parathion and gramine demonstrated negligible inhibition of the binding of both radioligands. Scatchard plots constructed from the inhibition of [3H]-oxotremorine-M binding in the absence and presence of different inhibitors showed an unaltered Bmax and a reduced affinity constant, indicative of potential competitive or allosteric mechanisms. The capacity of acetylcholinesterase inhibitors, with the exception of tacrine and ambenonium, to displace bound [3H]-oxotremorine-M in preference to [3H]quinuclinidyl benzilate predicts that the former compounds could act as potential agonists at muscarinic receptors. Moreover, the rank order for potency in inhibiting acetylcholinesterase (ambenonium>neostigmine=physostigmine =tacrine>pyridostigmine=edrophonium=galanthamine >desoxypeganine>parathion>gramine) indicated that the most effective inhibitors of acetylcholinesterase also displaced [3H]-oxotremorine-M to the greatest extent. The capacity of these inhibitors to displace [3H]-oxotremorine-M binding preclude their utilisation for the prevention of acetylcholine catabolism in rat brain membranes, the latter being required to estimate the binding of acetylcholine to [3H]-oxotremorine-M-labelled muscarinic receptors. However, fasciculin-2, a potent peptide inhibitor of acetylcholinesterase (IC50 24 nM), did prevent catabolism of acetylcholine in rat brain membranes with an atypical inhibition isotherm of [3H]-oxotremorine-M binding, thus permitting an estimation of the "global affinity" of acetylcholine (Ki 85 nM) for [3H]-oxotremorine-M-labelled muscarinic receptors in rat brain.

LHRH-pituitary plasma membrane binding: the presence of specific binding sites in other tissues.

PubMed

Marshall, J C; Shakespear, R A; Odell, W D

1976-11-01

Two specific binding sites for LHRH are present on plasma membranes prepared from rat and bovine anterior pituitary glands. One site is of high affinity (K = 2X108 1/MOL) and the second is of lower affinity (8-5X105 1/mol) and much greater capacity. Studies on membrane fractions prepared from other tissues showed the presence of a single specific site for LHRH. The kinetics and specificity of this site were similar to those of the lower affinity pituitary receptor. These results indicate that only pituitary membranes possess the higher affinity binding site and suggest that the low affinity site is not of physiological importance in the regulation of gonadotrophin secretion. After dissociation from membranes of non-pituitary tissues 125I-LHRH rebound to pituitary membrane preparations. Thus receptor binding per se does not result in degradation of LHRH and the function of these peripheral receptors remains obscure.

Synthesis and pharmacological evaluation of [(3)H]HS665, a novel, highly selective radioligand for the kappa opioid receptor.

PubMed

Guerrieri, Elena; Mallareddy, Jayapal Reddy; Tóth, Géza; Schmidhammer, Helmut; Spetea, Mariana

2015-03-18

Herein we report the radiolabeling and pharmacological investigation of a novel radioligand, the N-cyclobutylmethyl substituted diphenethylamine [(3)H]HS665, designed to bind selectively to the kappa opioid peptide (KOP) receptor, a target of therapeutic interest for the treatment of a variety of human disorders (i.e., pain, affective disorders, drug addiction, and psychotic disorders). HS665 was prepared in tritium-labeled form by a dehalotritiated method resulting in a specific activity of 30.65 Ci/mmol. Radioligand binding studies were performed to establish binding properties of [(3)H]HS665 to the recombinant human KOP receptor in membranes from Chinese hamster ovary cells stably expressing human KOP receptors (CHOhKOP) and to the native neuronal KOP receptor in guinea pig brain membranes. Binding of [(3)H]HS665 was specific and saturable in both tissue preparations. A single population of high affinity binding sites was labeled by [(3)H]HS665 in membranes from CHOhKOP cells and guinea pig brain with similar equilibrium dissociation constants, Kd, 0.45 and 0.64 nM, respectively. Average receptor density of [(3)H]HS665 recognition sites were 5564 and 154 fmol/mg protein in CHOhKOP cells and guinea pig brain, respectively. This study shows that the new radioligand distinguishes and labels KOP receptors specifically in neuronal and cellular systems expressing KOP receptors, making this molecule a valuable tool in probing structural and functional mechanisms governing ligand-KOP receptor interactions in both a recombinant and native in vitro setting.

Binding of [3H]MSX-2 (3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine) to rat striatal membranes--a new, selective antagonist radioligand for A(2A) adenosine receptors.

PubMed

Müller, C E; Maurinsh, J; Sauer, R

2000-01-01

The present study describes the preparation and binding properties of a new, potent, and selective A(2A) adenosine receptor (AR) antagonist radioligand, [3H]3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargy lxanth ine ([3H]MSX-2). [3H]MSX-2 binding to rat striatal membranes was saturable and reversible. Saturation experiments showed that [3H]MSX-2 labeled a single class of binding sites with high affinity (K(d)=8.0 nM) and limited capacity (B(max)=1.16 fmol.mg(-1) of protein). The presence of 100 microM GTP, or 10 mM magnesium chloride, respectively, had no effect on [3H]MSX-2 binding. AR agonists competed with the binding of 1 nM [3H]MSX-2 with the following order of potency: 5'-N-ethylcarboxamidoadenosine (NECA)>2-[4-(carboxyethyl)phenylethylamino]-5'-N-ethylcarboxami doaden osine (CGS-21680)>2-chloroadenosine (2-CADO)>N(6)-cyclopentyladenosine (CPA). AR antagonists showed the following order of potency: 8-(m-bromostyryl)-3, 7-dimethyl-1-propargylxanthine (BS-DMPX)>1, 3-dipropyl-8-cyclopentylxanthine (DPCPX)>(R)-5, 6-dimethyl-7-(1-phenylethyl)-2-(4-pyridyl)-7H-pyrrolo[2, 3-d]pyrimidine-4-amine (SH-128)>3,7-dimethyl-1-propargylxanthine (DMPX)>caffeine. The K(i) values for antagonists were in accordance with data from binding studies with the agonist radioligand [3H]CGS21680, while agonist affinities were 3-7-fold lower. [3H]MSX-2 is a highly selective A(2A) AR antagonist radioligand exhibiting a selectivity of at least two orders of magnitude versus all other AR subtypes. The new radioligand shows high specific radioactivity (85 Ci/mmol, 3150 GBq/mmol) and acceptable nonspecific binding at rat striatal membranes of 20-30%, at 1 nM.

The Receptor Binding Domain of Botulinum Neurotoxin Stereotype C Binds Phosphoinositides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yanfeng; Varnum, Susan M.

2012-03-01

Botulinum neurotoxins (BoNTs) are the most toxic proteins known for humans and animals with an extremely low LD50 of {approx} 1 ng/kg. BoNTs generally require a protein and a ganglioside on the cell membrane surface for binding, which is known as a 'dual receptor' mechanism for host intoxication. Recent studies have suggested that in addition to gangliosides, other membrane lipids such as phosphoinositides may be involved in the interactions with the receptor binding domain (HCR) of BoNTs for better membrane penetration. Here, using two independent lipid-binding assays, we tested the interactions of BoNT/C-HCR with lipids in vitro. BoNT/C-HCR was foundmore » to bind negatively charged phospholipids, preferentially phosphoinositides. Additional interactions to phosphoinositides may help BoNT/C bind membrane more tightly and transduct signals for subsequent steps of intoxication. Our results provide new insights into the mechanisms of host cell membrane recognition by BoNTs.« less

Tc-99m galactosyl-neoglycoalbumin: in vitro characterization of receptor-mediated binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vera, D.R.; Krohn, K.A.; Stadalnik, R.C.

1984-07-01

Hepatic binding protein (HBP) is a membrane receptor that binds and transports plasma glycoproteins from hepatic blood to hepatocellular lysosomes. A characterization is made of the in vitro binding of Tc-99m galactosyl-neoglycoalbumin (Tc-NGA), a synthetic HBP ligand, to liver membrane. Structural modifications of NGA resulted in the alteration of the equilibrium constant, KA, and the forward-binding rate constant, kb. Binding was second-order; the relative amount of membrane-bound NGA depended on the initial concentrations of ligand and membrane. Membrane displacement studies, using carrier ligands in contrast to previously bound Tc-NGA or I-NGA, correlated with the binding characteristics of a native HBPmore » ligand, asialo-orosomucoid. Computer simulation was used to study the detectability of the changes in HBP concentration at different values of kb. The simulations indicated that radiopharmacokinetic sensitivity to alterations in (HBP) should be possible using a neoglycoalbumin preparation with a carbohydrate density within the range of 15 to 25 galactose units per albumin molecule.« less

Isothermal titration calorimetric studies on the interaction of the major bovine seminal plasma protein, PDC-109 with phospholipid membranes.

PubMed

Anbazhagan, V; Sankhala, Rajeshwer S; Singh, Bhanu Pratap; Swamy, Musti J

2011-01-01

The interaction of the major bovine seminal plasma protein, PDC-109 with lipid membranes was investigated by isothermal titration calorimetry. Binding of the protein to model membranes made up of diacyl phospholipids was found to be endothermic, with positive values of binding enthalpy and entropy, and could be analyzed in terms of a single type of binding sites on the protein. Enthalpies and entropies for binding to diacylphosphatidylcholine membranes increased with increase in temperature, although a clear-cut linear dependence was not observed. The entropically driven binding process indicates that hydrophobic interactions play a major role in the overall binding process. Binding of PDC-109 with dimyristoylphosphatidylcholine membranes containing 25 mol% cholesterol showed an initial increase in the association constant as well as enthalpy and entropy of binding with increase in temperature, whereas the values decreased with further increase in temperature. The affinity of PDC-109 for phosphatidylcholine increased at higher pH, which is physiologically relevant in view of the basic nature of the seminal plasma. Binding of PDC-109 to Lyso-PC could be best analysed in terms of two types of binding interactions, a high affinity interaction with Lyso-PC micelles and a low-affinity interaction with the monomeric lipid. Enthalpy-entropy compensation was observed for the interaction of PDC-109 with phospholipid membranes, suggesting that water structure plays an important role in the binding process.

Isothermal Titration Calorimetric Studies on the Interaction of the Major Bovine Seminal Plasma Protein, PDC-109 with Phospholipid Membranes

PubMed Central

Anbazhagan, V.; Sankhala, Rajeshwer S.; Singh, Bhanu Pratap; Swamy, Musti J.

2011-01-01

The interaction of the major bovine seminal plasma protein, PDC-109 with lipid membranes was investigated by isothermal titration calorimetry. Binding of the protein to model membranes made up of diacyl phospholipids was found to be endothermic, with positive values of binding enthalpy and entropy, and could be analyzed in terms of a single type of binding sites on the protein. Enthalpies and entropies for binding to diacylphosphatidylcholine membranes increased with increase in temperature, although a clear-cut linear dependence was not observed. The entropically driven binding process indicates that hydrophobic interactions play a major role in the overall binding process. Binding of PDC-109 with dimyristoylphosphatidylcholine membranes containing 25 mol% cholesterol showed an initial increase in the association constant as well as enthalpy and entropy of binding with increase in temperature, whereas the values decreased with further increase in temperature. The affinity of PDC-109 for phosphatidylcholine increased at higher pH, which is physiologically relevant in view of the basic nature of the seminal plasma. Binding of PDC-109 to Lyso-PC could be best analysed in terms of two types of binding interactions, a high affinity interaction with Lyso-PC micelles and a low-affinity interaction with the monomeric lipid. Enthalpy-entropy compensation was observed for the interaction of PDC-109 with phospholipid membranes, suggesting that water structure plays an important role in the binding process. PMID:22022488

Viral membrane fusion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harrison, Stephen C., E-mail: harrison@crystal.harvard.edu

2015-05-15

Membrane fusion is an essential step when enveloped viruses enter cells. Lipid bilayer fusion requires catalysis to overcome a high kinetic barrier; viral fusion proteins are the agents that fulfill this catalytic function. Despite a variety of molecular architectures, these proteins facilitate fusion by essentially the same generic mechanism. Stimulated by a signal associated with arrival at the cell to be infected (e.g., receptor or co-receptor binding, proton binding in an endosome), they undergo a series of conformational changes. A hydrophobic segment (a “fusion loop” or “fusion peptide”) engages the target-cell membrane and collapse of the bridging intermediate thus formedmore » draws the two membranes (virus and cell) together. We know of three structural classes for viral fusion proteins. Structures for both pre- and postfusion conformations of illustrate the beginning and end points of a process that can be probed by single-virion measurements of fusion kinetics. - Highlights: • Viral fusion proteins overcome the high energy barrier to lipid bilayer merger. • Different molecular structures but the same catalytic mechanism. • Review describes properties of three known fusion-protein structural classes. • Single-virion fusion experiments elucidate mechanism.« less

Specific interaction of postsynaptic densities with membrane rafts isolated from synaptic plasma membranes.

PubMed

Liu, Qian; Yao, Wei-Dong; Suzuki, Tatsuo

2013-06-01

Postsynaptic membrane rafts are believed to play important roles in synaptic signaling, plasticity, and maintenance. We recently demonstrated the presence, at the electron microscopic level, of complexes consisting of membrane rafts and postsynaptic densities (PSDs) in detergent-resistant membranes (DRMs) prepared from synaptic plasma membranes (SPMs) ( Suzuki et al., 2011 , J Neurochem, 119, 64-77). To further explore these complexes, here we investigated the nature of the binding between purified SPM-DRMs and PSDs in vitro. In binding experiments, we used SPM-DRMs prepared after treating SPMs with n-octyl-β-d-glucoside, because at concentrations of 1.0% or higher it completely separates SPM-DRMs and PSDs, providing substantially PSD-free unique SPM-DRMs as well as DRM-free PSDs. PSD binding to PSD-free DRMs was identified by mass spectrometry, Western blotting, and electron microscopy. PSD proteins were not incorporated into SPMs, and significantly less PSD proteins were incorporated into DRMs prepared from liver membranes, providing in vitro evidence that binding of PSDs to DRMs is specific and suggestion of the presence of specific interacting molecules. These specific interactions may have important roles in synaptic development, function, and plasticity in vivo. In addition, the binding system we developed may be a good tool to search for binding molecules and binding mechanisms between PSDs and rafts.

Dynamics of Receptor-Mediated Nanoparticle Internalization into Endothelial Cells

PubMed Central

Gonzalez-Rodriguez, David; Barakat, Abdul I.

2015-01-01

Nanoparticles offer a promising medical tool for targeted drug delivery, for example to treat inflamed endothelial cells during the development of atherosclerosis. To inform the design of such therapeutic strategies, we develop a computational model of nanoparticle internalization into endothelial cells, where internalization is driven by receptor-ligand binding and limited by the deformation of the cell membrane and cytoplasm. We specifically consider the case of nanoparticles targeted against ICAM-1 receptors, of relevance for treating atherosclerosis. The model computes the kinetics of the internalization process, the dynamics of binding, and the distribution of stresses exerted between the nanoparticle and the cell membrane. The model predicts the existence of an optimal nanoparticle size for fastest internalization, consistent with experimental observations, as well as the role of bond characteristics, local cell mechanical properties, and external forces in the nanoparticle internalization process. PMID:25901833

The human Kell blood group binds the erythroid 4.1R protein: new insights into the 4.1R-dependent red cell membrane complex

PubMed Central

Azouzi, Slim; Collec, Emmanuel; Mohandas, Narla; An, Xiuli; Colin, Yves; Le Van Kim, Caroline

2015-01-01

Summary Protein 4.1R plays an important role in maintaining the mechanical properties of the erythrocyte membrane. We analysed the expression of Kell blood group protein in erythrocytes from a patient with hereditary elliptocytosis associated with complete 4.1R deficiency (4.1(−) HE). Flow cytometry and Western blot analyses revealed a severe reduction of Kell. In vitro pull down and co-immunoprecipitation experiments from erythrocyte membranes showed a direct interaction between Kell and 4.1R. Using different recombinant domains of 4.1R and the cytoplasmic domain of Kell, we demonstrated that the R46R motif in the juxta-membrane region of Kell binds to lobe B of the 4.1R FERM domain. We also observed that 4.1R deficiency is associated with a reduction of XK and DARC (also termed ACKR1) proteins, the absence of the glycosylated form of the urea transporter B and a slight decrease of band 3. The functional alteration of the 4.1(−) HE erythrocyte membranes was also determined by measuring various transport activities. We documented a slower rate of HCO3−/Cl− exchange, but normal water and ammonia transport across erythrocyte membrane in the absence of 4.1. These findings provide novel insights into the structural organization of blood group antigen proteins into the 4.1R complex of the human red cell membrane. PMID:26455906

Non-Native Metal Ion Reveals the Role of Electrostatics in Synaptotagmin 1-Membrane Interactions.

PubMed

Katti, Sachin; Nyenhuis, Sarah B; Her, Bin; Srivastava, Atul K; Taylor, Alexander B; Hart, P John; Cafiso, David S; Igumenova, Tatyana I

2017-06-27

C2 domains are independently folded modules that often target their host proteins to anionic membranes in a Ca 2+ -dependent manner. In these cases, membrane association is triggered by Ca 2+ binding to the negatively charged loop region of the C2 domain. Here, we used a non-native metal ion, Cd 2+ , in lieu of Ca 2+ to gain insight into the contributions made by long-range Coulombic interactions and direct metal ion-lipid bridging to membrane binding. Using X-ray crystallography, NMR, Förster resonance energy transfer, and vesicle cosedimentation assays, we demonstrate that, although Cd 2+ binds to the loop region of C2A/B domains of synaptotagmin 1 with high affinity, long-range Coulombic interactions are too weak to support membrane binding of individual domains. We attribute this behavior to two factors: the stoichiometry of Cd 2+ binding to the loop regions of the C2A and C2B domains and the impaired ability of Cd 2+ to directly coordinate the lipids. In contrast, electron paramagnetic resonance experiments revealed that Cd 2+ does support membrane binding of the C2 domains in full-length synaptotagmin 1, where the high local lipid concentrations that result from membrane tethering can partially compensate for lack of a full complement of divalent metal ions and specific lipid coordination in Cd 2+ -complexed C2A/B domains. Our data suggest that long-range Coulombic interactions alone can drive the initial association of C2A/B with anionic membranes and that Ca 2+ further augments membrane binding by the formation of metal ion-lipid coordination bonds and additional Ca 2+ ion binding to the C2 domain loop regions.

Interaction of Clostridium perfringens delta toxin with erythrocyte and liposome membranes and relation with the specific binding to the ganglioside GM2.

PubMed

Jolivet-Reynaud, C; Hauttecoeur, B; Alouf, J E

1989-01-01

The specific interaction of the cytolytic Clostridium perfringens delta toxin with membrane GM2 was indicated by: (i) characterization of this glycolipid in the membrane of sheep and goat erythrocytes, which are lysed by the toxin, whereas GM2 was undetectable in insensitive rabbit erythrocytes, (ii) demonstration of 125I-toxin binding to GM2, by autoradiography, following incubation with thin-layer chromatograms containing separated neuroblastoma gangliosides, and (iii) toxin fixation by phospholipid-cholesterol unilamellar vesicles containing either sheep gangliosides or GM2. In order to investigate the intramembrane events leading to membrane disruption following toxin binding, the photoreactive probe 12(4-azido-2-nitrophenoxy)stearoyl 1-14C glucosamine, which inserts into the outer layer and labels integral membrane proteins, was used to establish whether delta toxin penetrates into target cell membrane. No toxin labeling was found, suggesting that toxin action takes place at the membrane surface. This contention is supported by the observation that despite toxin binding, GM2 liposomes did not release entrapped 14C-glucose. Treatment of toxin with carboxypeptidases, but not aminopeptidases, abolished both toxin binding capacity onto erythrocytes and its combination with antitoxin neutralizing antibodies, suggesting that the carboxy terminal end of the toxin is critical for binding to cell membrane.

Anisotropic biodegradable lipid coated particles for spatially dynamic protein presentation.

PubMed

Meyer, Randall A; Mathew, Mohit P; Ben-Akiva, Elana; Sunshine, Joel C; Shmueli, Ron B; Ren, Qiuyin; Yarema, Kevin J; Green, Jordan J

2018-05-01

There has been growing interest in the use of particles coated with lipids for applications ranging from drug delivery, gene delivery, and diagnostic imaging to immunoengineering. To date, almost all particles with lipid coatings have been spherical despite emerging evidence that non-spherical shapes can provide important advantages including reduced non-specific elimination and increased target-specific binding. We combine control of core particle geometry with control of particle surface functionality by developing anisotropic, biodegradable ellipsoidal particles with lipid coatings. We demonstrate that these lipid coated ellipsoidal particles maintain advantageous properties of lipid polymer hybrid particles, such as the ability for modular protein conjugation to the particle surface using versatile bioorthogonal ligation reactions. In addition, they exhibit biomimetic membrane fluidity and demonstrate lateral diffusive properties characteristic of natural membrane proteins. These ellipsoidal particles simultaneously provide benefits of non-spherical particles in terms of stability and resistance to non-specific phagocytosis by macrophages as well as enhanced targeted binding. These biomaterials provide a novel and flexible platform for numerous biomedical applications. The research reported here documents the ability of non-spherical polymeric particles to be coated with lipids to form anisotropic biomimetic particles. In addition, we demonstrate that these lipid-coated biodegradable polymeric particles can be conjugated to a wide variety of biological molecules in a "click-like" fashion. This is of interest due to the multiple types of cellular mimicry enabled by this biomaterial based technology. These features include mimicry of the highly anisotropic shape exhibited by cells, surface presentation of membrane bound protein mimetics, and lateral diffusivity of membrane bound substrates comparable to that of a plasma membrane. This platform is demonstrated to facilitate targeted cell binding while being resistant to non-specific cellular uptake. Such a platform could allow for investigations into how physical parameters of a particle and its surface affect the interface between biomaterials and cells, as well as provide biomimetic technology platforms for drug delivery and cellular engineering. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Regulation of Hippocampal Glutamate Receptors: Evidence for the Involvement of a Calcium-Activated Protease

NASA Astrophysics Data System (ADS)

Baudry, Michel; Lynch, Gary

1980-04-01

Specific [3H]glutamate binding to rat hippocampal membranes and the calcium-induced increase in this binding are markedly temperature-sensitive and are inhibited by alkylating or reducing agents as well as by various protease inhibitors. N-Ethylmaleimide, chloromethyl ketone derivatives of lysine and phenylalanine, and tosylarginine methyl ester decrease the maximum number of [3H]glutamate binding sites without changing their affinity for glutamate. Preincubation of the membranes with glutamate does not protect the glutamate ``receptors'' from the suppressive effects of these agents. The proteases trypsin and α -chymotrypsin increase the maximum number of [3H]glutamate binding sites. The effects of calcium on glutamate binding are different across brain regions. Cerebellar membranes are almost insensitive whereas hippocampal and striatal membranes exhibit a strong increase in the number of binding sites after exposure to even low concentrations of calcium. These results suggest that an endogenous membrane-associated thiol protease regulates the number of [3H]glutamate binding sites in hippocampal membranes and that this is the mechanism by which calcium stimulates glutamate binding. The possibility is discussed that the postulated mechanisms participate in synaptic physiology and in particular may be related to the long-term potentiation of transmission found in hippocampus under certain conditions.

Ligand Binding Properties of the Lentil Lipid Transfer Protein: Molecular Insight into the Possible Mechanism of Lipid Uptake.

PubMed

Shenkarev, Zakhar O; Melnikova, Daria N; Finkina, Ekaterina I; Sukhanov, Stanislav V; Boldyrev, Ivan A; Gizatullina, Albina K; Mineev, Konstantin S; Arseniev, Alexander S; Ovchinnikova, Tatiana V

2017-03-28

The lentil lipid transfer protein, designated as Lc-LTP2, was isolated from Lens culinaris seeds. The protein belongs to the LTP1 subfamily and consists of 93 amino acid residues. Its spatial structure includes four α-helices (H1-H4) and a long C-terminal tail. Here, we report the ligand binding properties of Lc-LTP2. The fluorescent 2-p-toluidinonaphthalene-6-sulfonate binding assay revealed that the affinity of Lc-LTP2 for saturated and unsaturated fatty acids was enhanced with a decrease in acyl-chain length. Measurements of boundary potential in planar lipid bilayers and calcein dye leakage in vesicular systems revealed preferential interaction of Lc-LTP2 with the negatively charged membranes. Lc-LTP2 more efficiently transferred anionic dimyristoylphosphatidylglycerol (DMPG) than zwitterionic dimyristoylphosphatidylcholine. Nuclear magnetic resonance experiments confirmed the higher affinity of Lc-LTP2 for anionic lipids and those with smaller volumes of hydrophobic chains. The acyl chains of the bound lysopalmitoylphosphatidylglycerol (LPPG), DMPG, or dihexanoylphosphatidylcholine molecules occupied the internal hydrophobic cavity, while their headgroups protruded into the aqueous environment between helices H1 and H3. The spatial structure and backbone dynamics of the Lc-LTP2-LPPG complex were determined. The internal cavity was expanded from ∼600 to ∼1000 Å 3 upon the ligand binding. Another entrance into the internal cavity, restricted by the H2-H3 interhelical loop and C-terminal tail, appeared to be responsible for the attachment of Lc-LTP2 to the membrane or micelle surface and probably played an important role in the lipid uptake determining the ligand specificity. Our results confirmed the previous assumption regarding the membrane-mediated antimicrobial action of Lc-LTP2 and afforded molecular insight into its biological role in the plant.

Effects of bile salts on glucosylceramide containing membranes.

PubMed

Halin, Josefin; Mattjus, Peter

2011-12-01

The glycolipid transfer protein (GLTP) is capable of transporting glycolipids from a donor membrane, through the aqueous environment, to an acceptor membrane. The GLTP mediated glycolipid transfer from sphingomyelin membranes is very slow. In contrast, the transfer is fast from membranes composed of phosphatidylcholine. The lateral glycolipid membrane organization is known to be driven by their tendency to mix non-randomly with different membrane lipids. Consequently, the properties of the membrane lipids surrounding the glycolipids play an important role in the ability of GLTP to bind and transfer its substrates. Since GLTP transfer of glycolipids is almost nonexistent from sphingomyelin membranes, we have used this exceptionality to investigate if membrane intercalators can alter the membrane packing and induce glycolipid transfer. We found that the bile salts cholate, deoxycholate, taurocholate and taurodeoxycholate, cause glucosylceramide to become transferrable by GLTP. Other compounds, such as single chain lipids, ceramide and nonionic surfactants, that have membrane-perturbing effects, did not affect the transfer capability of GLTP. We speculate that the strong hydrogen bonding network formed in the interfacial region of glycosphingolipid-sphingomyelin membranes is disrupted by the membrane partition of the bile salts causing the glycosphingolipid to become transferrable. Copyright © 2011 Elsevier B.V. All rights reserved.

Dual inhibition of HY023016 based on binding properties of platelet membrane receptor subunit glycoprotein Ibα and thrombin exosites.

PubMed

Chen, Qiu-Fang; Cui, Shuang; Shen, Hui-Liang; Chen, Xiang; Li, Yun-Zhan; Wu, Qian; Xu, Yun-Gen; Gong, Guo-Qing

2018-03-05

Thrombin has long been suggested as a desirable antithrombotic target, but anti-thrombin therapy without anti-platelet thereby has never achieved the ideal effect. HY023016 is a novel compound, in our previous study, it exerted better anti-thrombotic than dabigatran etexilate. The present study aims to illustrate the excess anti-thrombotic molecular mechanisms of HY023016 through thrombin anion exosites and the platelet membrane receptor subunit glycoprotein Ibα (GPIbα). HY023016 strongly inhibited the conversion of fibrinogen to fibrous may via blocking thrombin exosite I. We also discovered that HY023016 remarkably inhibited exosite II by a loss of affinity for the γ'-peptide of fibrinogen and for heparin. Furthermore, a solid phase binding assay revealed that HY023016 inhibited ristocetin-induced washed platelets bind to von Willebrand factor (vWF). In GST pull-down assay, HY023016 decreased the binding of recombinant vWF-A1 to GPIbα N-terminal. Thus, HY023016 provides an innovative idea for designing multi-targeted anti-thrombotic drugs and laying a scientific foundation for reducing "total thrombosis risk" in a clinical drug treatment. Copyright © 2018 Elsevier B.V. All rights reserved.

A novel-type phosphatidylinositol phosphate-interactive, Ca-binding protein PCaP1 in Arabidopsis thaliana: stable association with plasma membrane and partial involvement in stomata closure.

PubMed

Nagata, Chisako; Miwa, Chika; Tanaka, Natsuki; Kato, Mariko; Suito, Momoe; Tsuchihira, Ayako; Sato, Yori; Segami, Shoji; Maeshima, Masayoshi

2016-05-01

The Ca(2+)-binding protein-1 (PCaP1) of Arabidopsis thaliana is a new type protein that binds to phosphatidylinositol phosphates and Ca(2+)-calmodulin complex as well as free Ca(2+). Although biochemical properties, such as binding to ligands and N-myristoylation, have been revealed, the intracellular localization, tissue and cell specificity, integrity of membrane association and physiological roles of PCaP1 are unknown. We investigated the tissue and intracellular distribution of PCaP1 by using transgenic lines expressing PCaP1 linked with a green fluorescence protein (GFP) at the carboxyl terminus of PCaP1. GFP fluorescence was obviously detected in most tissues including root, stem, leaf and flower. In these tissues, PCaP1-GFP signal was observed predominantly in the plasma membrane even under physiological stress conditions but not in other organelles. The fluorescence was detected in the cytosol when the 25-residue N-terminal sequence was deleted from PCaP1 indicating essential contribution of N-myristoylation to the plasma membrane anchoring. Fluorescence intensity of PCaP1-GFP in roots was slightly decreased in seedlings grown in medium supplemented with high concentrations of iron for 1 week and increased in those grown with copper. In stomatal guard cells, PCaP1-GFP was strictly, specifically localized to the plasma membrane at the epidermal-cell side but not at the pore side. A T-DNA insertion mutant line of PCaP1 did not show marked phenotype in a life cycle except for well growth under high CO2 conditions. However, stomata of the mutant line did not close entirely even in high osmolarity, which usually induces stomata closure. These results suggest that PCaP1 is involved in the stomatal movement, especially closure process, in leaves and response to excessive copper in root and leaf as a mineral nutrient as a physiological role.

Insights into the Membrane Interactions of the Saposin-Like Proteins Na-SLP-1 and Ac-SLP-1 from Human and Dog Hookworm

PubMed Central

Willis, Charlene; Wang, Conan K.; Osman, Asiah; Simon, Anne; Pickering, Darren; Mulvenna, Jason; Riboldi-Tunicliffe, Alan; Jones, Malcolm K.; Loukas, Alex; Hofmann, Andreas

2011-01-01

Saposin-like proteins (SAPLIPs) from soil-transmitted helminths play pivotal roles in host-pathogen interactions and have a high potential as targets for vaccination against parasitic diseases. We have identified two non-orthologous SAPLIPs from human and dog hookworm, Na-SLP-1 and Ac-SLP-1, and solved their three-dimensional crystal structures. Both proteins share the property of membrane binding as monitored by liposome co-pelleting assays and monolayer adsorption. Neither SAPLIP displayed any significant haemolytic or bactericidal activity. Based on the structural information, as well as the results from monolayer adsorption, we propose models of membrane interactions for both SAPLIPs. Initial membrane contact of the monomeric Na-SLP-1 is most likely by electrostatic interactions between the membrane surface and a prominent basic surface patch. In case of the dimeric Ac-SLP-1, membrane interactions are most likely initiated by a unique tryptophan residue that has previously been implicated in membrane interactions in other SAPLIPs. PMID:21991310

Insights into the membrane interactions of the saposin-like proteins Na-SLP-1 and Ac-SLP-1 from human and dog hookworm.

PubMed

Willis, Charlene; Wang, Conan K; Osman, Asiah; Simon, Anne; Pickering, Darren; Mulvenna, Jason; Riboldi-Tunicliffe, Alan; Jones, Malcolm K; Loukas, Alex; Hofmann, Andreas

2011-01-01

Saposin-like proteins (SAPLIPs) from soil-transmitted helminths play pivotal roles in host-pathogen interactions and have a high potential as targets for vaccination against parasitic diseases. We have identified two non-orthologous SAPLIPs from human and dog hookworm, Na-SLP-1 and Ac-SLP-1, and solved their three-dimensional crystal structures. Both proteins share the property of membrane binding as monitored by liposome co-pelleting assays and monolayer adsorption. Neither SAPLIP displayed any significant haemolytic or bactericidal activity. Based on the structural information, as well as the results from monolayer adsorption, we propose models of membrane interactions for both SAPLIPs. Initial membrane contact of the monomeric Na-SLP-1 is most likely by electrostatic interactions between the membrane surface and a prominent basic surface patch. In case of the dimeric Ac-SLP-1, membrane interactions are most likely initiated by a unique tryptophan residue that has previously been implicated in membrane interactions in other SAPLIPs.

β-chain of ATP synthase as a lipophorin binding protein and its role in lipid transfer in the midgut of Panstrongylus megistus (Hemiptera: Reduviidae).

PubMed

Fruttero, Leonardo L; Demartini, Diogo R; Rubiolo, Edilberto R; Carlini, Célia R; Canavoso, Lilián E

2014-09-01

Lipophorin, the main lipoprotein in the circulation of the insects, cycles among peripheral tissues to exchange its lipid cargo at the plasma membrane of target cells, without synthesis or degradation of its apolipoprotein matrix. Currently, there are few characterized candidates supporting the functioning of the docking mechanism of lipophorin-mediated lipid transfer. In this work we combined ligand blotting assays and tandem mass spectrometry to characterize proteins with the property to bind lipophorin at the midgut membrane of Panstrongylus megistus, a vector of Chagas' disease. We further evaluated the role of lipophorin binding proteins in the transfer of lipids between the midgut and lipophorin. The β subunit of the ATP synthase complex (β-ATPase) was identified as a lipophorin binding protein. β-ATPase was detected in enriched midgut membrane preparations free of mitochondria. It was shown that β-ATPase partially co-localizes with lipophorin at the plasma membrane of isolated enterocytes and in the sub-epithelial region of the midgut tissue. The interaction of endogenous lipophorin and β-ATPase was also demonstrated by co-immunoprecipitation assays. Blocking of β-ATPase significantly diminished the binding of lipophorin to the isolated enterocytes and to the midgut tissue. In vivo assays injecting the β-ATPase antibody significantly reduced the transfer of [(3)H]-diacylglycerol from the midgut to the hemolymph in insects fed with [9,10-(3)H]-oleic acid, supporting the involvement of lipophorin-β-ATPase association in the transfer of lipids. In addition, the β-ATPase antibody partially impaired the transfer of fatty acids from lipophorin to the midgut, a less important route of lipid delivery to this tissue. Taken together, the findings strongly suggest that β-ATPase plays a role as a docking lipophorin receptor at the midgut of P. megistus. Copyright © 2014 Elsevier Ltd. All rights reserved.

Membrane-Mediated Cooperativity of Proteins

NASA Astrophysics Data System (ADS)

Weikl, Thomas R.

2018-04-01

Besides direct protein-protein interactions, indirect interactions mediated by membranes play an important role for the assembly and cooperative function of proteins in membrane shaping and adhesion. The intricate shapes of biological membranes are generated by proteins that locally induce membrane curvature. Indirect curvature-mediated interactions between these proteins arise because the proteins jointly affect the bending energy of the membranes. These curvature-mediated interactions are attractive for crescent-shaped proteins and are a driving force in the assembly of the proteins during membrane tubulation. Membrane adhesion results from the binding of receptor and ligand proteins that are anchored in the apposing membranes. The binding of these proteins strongly depends on nanoscale shape fluctuations of the membranes, leading to a fluctuation-mediated binding cooperativity. A length mismatch between receptor-ligand complexes in membrane adhesion zones causes repulsive curvature-mediated interactions that are a driving force for the length-based segregation of proteins during membrane adhesion.

3- and 4-O-sulfoconjugated and methylated dopamine: highly reduced binding affinity to dopamine D2 receptors in rat striatal membranes.

PubMed

Werle, E; Lenz, T; Strobel, G; Weicker, H

1988-07-01

The binding properties of 3- and 4-O-sulfo-conjugated dopamine (DA-3-O-S, DA-4-O-S) as well as 3-O-methylated dopamine (MT) to rat striatal dopamine D2 receptors were investigated. 3H-spiperone was used as a radioligand in the binding studies. In saturation binding experiments (+)butaclamol, which has been reported to bind to dopaminergic D2 and serotoninergic 5HT2 receptors, was used in conjunction with ketanserin and sulpiride, which preferentially label 5HT2 and D2 receptors, respectively, in order to discriminate between 3H-spiperone binding to D2 and to 5HT2 receptors. Under our particular membrane preparation and assay conditions, 3H-spiperone binds to D2 and 5HT2 receptors with a maximal binding capacity (Bmax) of 340 fmol/mg protein in proportions of about 75%:25% with similar dissociation constants KD (35 pmol/l; 43 pmol/l). This result was verified by the biphasic competition curve of ketanserin, which revealed about 20% high (KD = 24 nmol/l) and 80% low (KD = 420 nmol/l) affinity binding sites corresponding to 5HT2 and D2 receptors, respectively. Therefore, all further competition experiments at a tracer concentration of 50 pmol/l were performed in the presence of 0.1 mumol/l ketanserin to mask the 5HT2 receptors. DA competition curves were best fitted assuming two binding sites, with high (KH = 0.12 mumol/l) and low (KL = 18 mumol/l) affinity, present in a ratio of 3:1. The high affinity binding sites were interconvertible by 100 mumol/l guanyl-5-yl imidodiphosphate [Gpp(NH)p], resulting in a homogenous affinity state of DA receptors (KD = 2.8 mumol/l).2+ off

[3H]aniracetam binds to specific recognition sites in brain membranes.

PubMed

Fallarino, F; Genazzani, A A; Silla, S; L'Episcopo, M R; Camici, O; Corazzi, L; Nicoletti, F; Fioretti, M C

1995-08-01

[3H]Aniracetam bound to specific and saturable recognition sites in membranes prepared from discrete regions of rat brain. In crude membrane preparation from rat cerebral cortex, specific binding was Na+ independent, was still largely detectable at low temperature (4 degrees C), and underwent rapid dissociation. Scatchard analysis of [3H]aniracetam binding revealed a single population of sites with an apparent KD value of approximately 70 nM and a maximal density of 3.5 pmol/mg of protein. Specifically bound [3H]aniracetam was not displaced by various metabolites of aniracetam, nor by other pyrrolidinone-containing nootropic drugs such as piracetam or oxiracetam. Subcellular distribution studies showed that a high percentage of specific [3H]aniracetam binding was present in purified synaptosomes or mitochondria, whereas specific binding was low in the myelin fraction. The possibility that at least some [3H]aniracetam binding sites are associated with glutamate receptors is supported by the evidence that specific binding was abolished when membranes were preincubated at 37 degrees C under fast shaking (a procedure that substantially reduced the amount of glutamate trapped in the membranes) and could be restored after addition of either glutamate or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) but not kainate. The action of AMPA was antagonized by DNQX, which also reduced specific [3H]aniracetam binding in unwashed membranes. High levels of [3H]aniracetam binding were detected in hippocampal, cortical, or cerebellar membranes, which contain a high density of excitatory amino acid receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Surface plasmon resonance spectroscopy for characterisation of membrane protein-ligand interactions and its potential for drug discovery.

PubMed

Patching, Simon G

2014-01-01

Surface plasmon resonance (SPR) spectroscopy is a rapidly developing technique for the study of ligand binding interactions with membrane proteins, which are the major molecular targets for validated drugs and for current and foreseeable drug discovery. SPR is label-free and capable of measuring real-time quantitative binding affinities and kinetics for membrane proteins interacting with ligand molecules using relatively small quantities of materials and has potential to be medium-throughput. The conventional SPR technique requires one binding component to be immobilised on a sensor chip whilst the other binding component in solution is flowed over the sensor surface; a binding interaction is detected using an optical method that measures small changes in refractive index at the sensor surface. This review first describes the basic SPR experiment and the challenges that have to be considered for performing SPR experiments that measure membrane protein-ligand binding interactions, most importantly having the membrane protein in a lipid or detergent environment that retains its native structure and activity. It then describes a wide-range of membrane protein systems for which ligand binding interactions have been characterised using SPR, including the major drug targets G protein-coupled receptors, and how challenges have been overcome for achieving this. Finally it describes some recent advances in SPR-based technology and future potential of the technique to screen ligand binding in the discovery of drugs. This article is part of a Special Issue entitled: Structural and biophysical characterisation of membrane protein-ligand binding. Copyright © 2013 Elsevier B.V. All rights reserved.

Repair of Nerve Cell Membrance Damage by Calcium-Dependent, Membrane-Binding Proteins

DTIC Science & Technology

2013-09-01

In acute spinal cord injury the plasma membranes of spinal neurons are torn allowing high concentrations of calcium to enter the cytoplasm, activating...repairing the cell membrane as soon as the increase in intracellular calcium is sensed by calcium -binding proteins. If these repair mechanisms can be...testing the hypothesis that the action of copine, a human calcium -dependent-membrane-binding protein, in model systems can promote a stable repair of

Inhibition of vincristine binding to plasma membrane vesicles from daunorubicin-resistant Ehrlich ascites cells by multidrug resistance modulators.

PubMed Central

Sehested, M.; Jensen, P. B.; Skovsgaard, T.; Bindslev, N.; Demant, E. J.; Friche, E.; Vindeløv, L.

1989-01-01

The multidrug resistance (MDR) phenotype is presumed to be mostly dependent on changes in the resistant cell plasma membrane, notably the emergence of a 170 kDa glycoprotein called P-glycoprotein, which facilitate increased drug efflux. We have previously demonstrated that ATP-enhanced binding of vincristine (VCR) to plasma membrane vesicles is much greater in MDR than in wild type cells. The present study has shown that VCR binding to MDR Ehrlich ascites tumour cell plasma membrane vesicles is inhibited 50% most efficiently by quinidine (0.5 microM) followed by verapamil (4.1 microM) and trifluoperazine (23.2 microM). This is the reverse order of the effect on whole cells where a ranking of efficiency in terms of enhancement of VCR accumulation, inhibition of VCR efflux, DNA perturbation and modulation of resistance in a clonogenic assay, was trifluoperazine greater than or equal to verapamil much greater than quinidine. The detergent Tween 80 inhibited VCR binding to plasma membrane vesicles at 0.001% v/v which agreed with the level which modulated resistance and increased VCR accumulation in whole cells. No effect was observed on daunorubicin binding to MDR plasma membrane vesicles after incubation with either Tween 80 (up to 0.1% v/v) or verapamil (up to 25 microM). We conclude that the effect of a modulating drug in reversing resistance to VCR correlates with its ability to raise intracellular VCR levels but not with its capability to inhibit VCR binding to the plasma membrane. Thus, enhancement of VCR accumulation in MDR cells is hardly solely due to competition for a drug binding site on P-glycoprotein. Furthermore, the lack of a demonstrable effect on daunorubicin binding to the plasma membrane by modulators points to transport mechanisms which do not utilise specific drug binding to the plasma membrane. PMID:2605092

Molecular mechanism of membrane binding of the GRP1 PH domain.

PubMed

Lai, Chun-Liang; Srivastava, Anand; Pilling, Carissa; Chase, Anna R; Falke, Joseph J; Voth, Gregory A

2013-09-09

The pleckstrin homology (PH) domain of the general receptor of phosphoinositides 1 (GRP1) protein selectively binds to a rare signaling phospholipid, phosphatidylinositol (3,4,5)-trisphosphate (PIP3), in the membrane. The specific PIP3 lipid docking of GRP1 PH domain is essential to protein cellular function and is believed to occur in a stepwise process, electrostatic-driven membrane association followed by the specific PIP3 binding. By a combination of all-atom molecular dynamics (MD) simulations, coarse-grained analysis, electron paramagnetic resonance (EPR) membrane docking geometry, and fluorescence resonance energy transfer (FRET) kinetic studies, we have investigated the search and bind process in the GRP1 PH domain at the molecular scale. We simulated the two membrane binding states of the GRP1 PH domain in the PIP3 search process, before and after the GRP1 PH domain docks with the PIP3 lipid. Our results suggest that the background anionic phosphatidylserine lipids, which constitute around one-fifth of the membrane by composition, play a critical role in the initial stages of recruiting protein to the membrane surface through non-specific electrostatic interactions. Our data also reveal a previously unseen transient membrane association mechanism that is proposed to enable a two-dimensional "hopping" search of the membrane surface for the rare PIP3 target lipid. We further modeled the PIP3-bound membrane-protein system using the EPR membrane docking structure for the MD simulations, quantitatively validating the EPR membrane docking structure and augmenting our understanding of the binding interface with atomic-level detail. Several observations and hypotheses reached from our MD simulations are also supported by experimental kinetic studies. Copyright © 2013 Elsevier Ltd. All rights reserved.

Amphipathic alpha-helices and putative cholesterol binding domains of the influenza virus matrix M1 protein are crucial for virion structure organisation.

PubMed

Tsfasman, Tatyana; Kost, Vladimir; Markushin, Stanislav; Lotte, Vera; Koptiaeva, Irina; Bogacheva, Elena; Baratova, Ludmila; Radyukhin, Victor

2015-12-02

The influenza virus matrix M1 protein is an amphitropic membrane-associated protein, forming the matrix layer immediately beneath the virus raft membrane, thereby ensuring the proper structure of the influenza virion. The objective of this study was to elucidate M1 fine structural characteristics, which determine amphitropic properties and raft membrane activities of the protein, via 3D in silico modelling with subsequent mutational analysis. Computer simulations suggest the amphipathic nature of the M1 α-helices and the existence of putative cholesterol binding (CRAC) motifs on six amphipathic α-helices. Our finding explains for the first time many features of this protein, particularly the amphitropic properties and raft/cholesterol binding potential. To verify these results, we generated mutants of the A/WSN/33 strain via reverse genetics. The M1 mutations included F32Y in the CRAC of α-helix 2, W45Y and W45F in the CRAC of α-helix 3, Y100S in the CRAC of α-helix 6, M128A and M128S in the CRAC of α-helix 8 and a double L103I/L130I mutation in both a putative cholesterol consensus motif and the nuclear localisation signal. All mutations resulted in viruses with unusual filamentous morphology. Previous experimental data regarding the morphology of M1-gene mutant influenza viruses can now be explained in structural terms and are consistent with the pivotal role of the CRAC-domains and amphipathic α-helices in M1-lipid interactions. Copyright © 2015 Elsevier B.V. All rights reserved.

Controlling Cargo Trafficking in Multicomponent Membranes.

PubMed

Curk, Tine; Wirnsberger, Peter; Dobnikar, Jure; Frenkel, Daan; Šarić, Anđela

2018-04-27

Biological membranes typically contain a large number of different components dispersed in small concentrations in the main membrane phase, including proteins, sugars, and lipids of varying geometrical properties. Most of these components do not bind the cargo. Here, we show that such "inert" components can be crucial for the precise control of cross-membrane trafficking. Using a statistical mechanics model and molecular dynamics simulations, we demonstrate that the presence of inert membrane components of small isotropic curvatures dramatically influences cargo endocytosis, even if the total spontaneous curvature of such a membrane remains unchanged. Curved lipids, such as cholesterol, as well as asymmetrically included proteins and tethered sugars can, therefore, actively participate in the control of the membrane trafficking of nanoscopic cargo. We find that even a low-level expression of curved inert membrane components can determine the membrane selectivity toward the cargo size and can be used to selectively target membranes of certain compositions. Our results suggest a robust and general method of controlling cargo trafficking by adjusting the membrane composition without needing to alter the concentration of receptors or the average membrane curvature. This study indicates that cells can prepare for any trafficking event by incorporating curved inert components in either of the membrane leaflets.

Membrane-Protein Binding Measured with Solution-Phase Plasmonic Nanocube Sensors

PubMed Central

Wu, Hung-Jen; Henzie, Joel; Lin, Wan-Chen; Rhodes, Christopher; Li, Zhu; Sartorel, Elodie; Thorner, Jeremy; Yang, Peidong; Groves, Jay. T.

2013-01-01

We describe a solution-phase sensor of lipid-protein binding based on localized surface plasmon resonance (LSPR) of silver nanocubes. When silica-coated nanocubes are mixed into a suspension of lipid vesicles, supported membranes spontaneously assemble on their surfaces. Using a standard laboratory spectrophotometer, we calibrate the LSPR peak shift due to protein binding to the membrane surface and then characterize the lipid-binding specificity of a pleckstrin-homology domain protein. PMID:23085614

Differences in Organizational Structure of Insulin Receptor on Rat Adipocyte and Liver Plasma Membranes: Role of Disulfide Bonds

NASA Astrophysics Data System (ADS)

Schweitzer, John B.; Smith, Robert M.; Jarett, Leonard

1980-08-01

Binding of 125I-labeled insulin to rat liver and adipocyte plasma membranes has been investigated after treatment of the membranes with agents that modify disulfide bonds or sulfhydryl groups. Dithiothreitol, a disulfide-reducing agent, produced a bimodal response in adipocyte plasma membranes with dose-dependent increases in binding occurring over the range of 0-1 mM dithiothreitol; 5 mM dithiothreitol produced decreased binding. Insulin binding reached its maximal increase at 1 mM and was 3 times control values. Scatchard analysis of the 1 mM dithiothreitol effect revealed a straight line plot indicative of one class of sites with a Ka of 1.0× 108 M-1 which is intermediate between the two Kas obtained from the curvilinear Scatchard plot of control membranes. There was a 20-fold increase in the number of intermediate-affinity receptors compared to high-affinity receptors. The increased 125I-labeled insulin binding after dithiothreitol treatment was reversed by oxidized glutathione in a dose-dependent manner. Interposition of treatment with N-ethylmaleimide, an alkylating agent, prevented oxidized glutathione from reversing the dithiothreitol effect. Reduced glutathione produced the same effect as dithiothreitol. Liver plasma membranes treated with up to 1 mM dithiothreitol exhibited a maximum increase in insulin binding of 20% compared to control. Dithiothreitol at 5 mM decreased insulin binding below that of control membranes. The results indicate that the dithiothreitol effect on insulin binding to adipocyte plasma membranes is due to disruption of disulfide bonds, and that the structural organization of the insulin receptor on the plasma membranes is different for liver and for adipose tissue. The data imply that the insulin receptors on the plasma membrane of adipocytes possess at least two functionally distinct subclasses of disulfide bond but liver insulin receptors do not.

Synthesis and Properties of Dodecyl Trehaloside Detergents for Membrane Protein Studies

PubMed Central

Tao, Houchao; Fu, Yu; Thompson, Aaron; Lee, Sung Chang; Mahoney, Nicholas; Stevens, Raymond C.; Zhang, Qinghai

2012-01-01

Sugar-based detergents, mostly derived from maltose or glucose, prevail in the extraction, solubilization, stabilization and crystallization of membrane proteins. Inspired by the broad use of trehalose for protecting biological macromolecules and lipid bilayer structures, we synthesized new trehaloside detergents for potential applications in membrane protein research. We devised an efficient synthesis of four dodecyl trehalosides, each with the 12-carboned alkyl chain attached to different hydroxyl groups of trehalose, thus presenting a structurally diverse but related family of detergents. The detergent physical properties, including solubility, hydrophobicity, critical micelle concentration (CMC) and size of micelles, were evaluated and compared with the most popular maltoside analog, β- D-dodecylmaltoside (DDM), which varied from each other due to distinct molecular geometries and possible polar group interactions in resulting micelles. Crystals of 2-dodecyl trehaloside (2-DDTre) were also obtained in methanol, and the crystal packing revealed multiple H-bonded interactions among adjacent trehalose groups. The few trehaloside detergents were tested for the solubilization and stabilization of the nociceptin/orphanin FQ peptide receptor (ORL1) and MsbA, which belong to the G-protein coupled receptor (GPCR) and ATP-binding cassette transporter families, respectively. Our results demonstrated the utility of trehaloside detergents as membrane protein solubilization reagents with the optimal detergents being protein dependent. Continuing development and investigations of trehaloside detergents are attractive given their interesting and unique chemical-physical properties and potential interactions with membrane lipids. PMID:22780816

Inhibition of Oncogenic functionality of STAT3 Protein by Membrane Anchoring

NASA Astrophysics Data System (ADS)

Liu, Baoxu; Fletcher, Steven; Gunning, Patrick; Gradinaru, Claudiu

2009-03-01

Signal Transducer and Activator of Transcription 3 (STAT3) protein plays an important role in oncogenic processes. A novel molecular therapeutic approach to inhibit the oncogenic functionality of STAT3 is to design a prenylated small peptide sequence which could sequester STAT3 to the plasma membrane. We have also developed a novel fluorescein derivative label (F-NAc), which is much more photostable compared to the popular fluorescein label FITC. Remarkably, the new dye shows fluorescent properties that are invariant over a wide pH range, which is advantageous for our application. We have shown that F-NAc is suitable for single-molecule measurements and its properties are not affected by ligation to biomolecules. The membrane localization via high-affinity prenylated small-molecule binding agents is studied by encapsulating FNAc-labeled STAT3 and inhibitors within a liposome model cell system. The dynamics of the interaction between the protein and the prenylated ligands is investigated at single molecule level. The efficiency and stability of the STAT3 anchoring in lipid membranes are addressed via quantitative confocal imaging and single-molecule spectroscopy using a custom-built multiparameter fluorescence microscope.

Modulation of physiological and pathological activities of lysozyme by biological membranes.

PubMed

Trusova, Valeriya

2012-09-01

The molecular details of interactions between lipid membranes and lysozyme (Lz), a small polycationic protein with a wide range of biological activities, have long been the focus of numerous studies. The biological consequences of this process are considered to embrace at least two aspects: i) correlation between antimicrobial and membranotropic properties of this protein, and ii) lipid-mediated Lz amyloidogenesis. The mechanisms underlying the lipid-assisted protein fibrillogenesis and membrane disruption exerted by Lz in bacterial cells are believed to be similar. The present investigation was undertaken to gain further insight into Lz-lipid interactions and explore the routes by which Lz exerts its antimicrobial and amyloidogenic actions. Binding and Förster resonance energy transfer studies revealed that upon increasing the content of anionic lipids in lipid vesicles, Lz forms aggregates in a membrane environment. Total internal reflection fluorescence microscopy and pyrene excimerization reaction were employed to study the effect of Lz on the structural and dynamic properties of lipid bilayers. It was found that Lz induces lipid demixing and reduction of bilayer free volume, the magnitude of this effect being much more pronounced for oligomeric protein.

Binding properties of Clostridium botulinum type C progenitor toxin to mucins.

PubMed

Nakamura, Toshio; Takada, Noriko; Tonozuka, Takashi; Sakano, Yoshiyuki; Oguma, Keiji; Nishikawa, Atsushi

2007-04-01

It has been reported that Clostridium botulinum type C 16S progenitor toxin (C16S toxin) first binds to the sialic acid on the cell surface of mucin before invading cells [A. Nishikawa, N. Uotsu, H. Arimitsu, J.C. Lee, Y. Miura, Y. Fujinaga, H. Nakada, T. Watanabe, T. Ohyama, Y. Sakano, K. Oguma, The receptor and transporter for internalization of Clostridium botulinum type C progenitor toxin into HT-29 cells, Biochem. Biophys. Res. Commun. 319 (2004) 327-333]. In this study we investigated the binding properties of the C16S toxin to glycoproteins. Although the toxin bound to membrane blotted mucin derived from the bovine submaxillary gland (BSM), which contains a lot of sialyl oligosaccharides, it did not bind to neuraminidase-treated BSM. The binding of the toxin to BSM was inhibited by N-acetylneuraminic acid, N-glycolylneuraminic acid, and sialyl oligosaccharides strongly, but was not inhibited by neutral oligosaccharides. Both sialyl alpha2-3 lactose and sialyl alpha2-6 lactose prevented binding similarly. On the other hand, the toxin also bound well to porcine gastric mucin. In this case, neutral oligosaccharides might play an important role as ligand, since galactose and lactose inhibited binding. These results suggest that the toxin is capable of recognizing a wide variety of oligosaccharide structures.

Liposome Disruption Assay to Examine Lytic Properties of Biomolecules.

PubMed

Jimah, John R; Schlesinger, Paul H; Tolia, Niraj H

2017-08-05

Proteins may have three dimensional structural or amino acid features that suggest a role in targeting and disrupting lipids within cell membranes. It is often necessary to experimentally investigate if these proteins and biomolecules are able to disrupt membranes in order to conclusively characterize the function of these biomolecules. Here, we describe an in vitro assay to evaluate the membrane lytic properties of proteins and biomolecules. Large unilamellar vesicles (liposomes) containing carboxyfluorescein at fluorescence-quenching concentrations are treated with the biomolecule of interest. A resulting increase in fluorescence due to leakage of the dye from liposomes and subsequent dilution in the buffer demonstrates that the biomolecule is sufficient for disrupting liposomes and membranes. Additionally, since liposome disruption may occur via pore-formation or via general solubilization of lipids similar to detergents, we provide a method to distinguish between these two mechanisms. Pore-formation can be identified and evaluated by examining the blockade of carboxyfluorescein release with dextran molecules that fit the pore. The methods described here were used to determine that the malaria vaccine candidate CelTOS and proapoptotic Bax disrupt liposomes by pore formation (Saito et al. , 2000; Jimah et al. , 2016). Since membrane lipid binding by a biomolecule precedes membrane disruption, we recommend the companion protocol: Jimah et al. , 2017.

Autoinhibition of ankyrin-B/G membrane target bindings by intrinsically disordered segments from the tail regions

PubMed Central

Wang, Chao; Wei, Zhiyi

2017-01-01

Ankyrins together with their spectrin partners are the master organizers of micron-scale membrane domains in diverse tissues. The 24 ankyrin (ANK) repeats of ankyrins bind to numerous membrane proteins, linking them to spectrin-based cytoskeletons at specific membrane microdomains. The accessibility of the target binding groove of ANK repeats must be regulated to achieve spatially defined functions of ankyrins/target complexes in different tissues, though little is known in this regard. Here we systemically investigated the autoinhibition mechanism of ankyrin-B/G by combined biochemical, biophysical and structural biology approaches. We discovered that the entire ANK repeats are inhibited by combinatorial and quasi-independent bindings of multiple disordered segments located in the ankyrin-B/G linkers and tails, suggesting a mechanistic basis for differential regulations of membrane target bindings by ankyrins. In addition to elucidating the autoinhibition mechanisms of ankyrins, our study may also shed light on regulations on target bindings by other long repeat-containing proteins. PMID:28841137

[Analysis of the binding capacity of the benzodiazepine site of gabaa receptor in mice C57BL/6 and BALB/C pretreated with anxiolytics].

PubMed

Iarkova, M A

2011-01-01

The level of specific 3H-flunitrazepam binding in synaptosomal membranes of C57BL/6 and BALB/c mice brain underwent to the stress of different types has been studied. Mild stress (Elevated Plus Maze) was shown to induce the decrease of benzodiazepine binding in BALB/c mice only, while the strong one (Exposure to a predator) was revealed to cause this decrease in both strains. Behavioral effects of different non-benzodiazepine drugs possessing anxiolytic properties (Afobazol, Ladasten and Noopept) was accompanied with the normalization of the level of benzodiazepine reception, reduced by the stress of both modalities.

Isolation and characterization of a Ca/sup 2 +/ carrier candidate from calf heart inner mitochondrial membrane

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jeng, A.Y.

1979-01-01

A protein was isolated from calf heart inner mitochondrial membrane with the aid of an electron paramagnetic resonance assay based on the relative binding properties of Ca/sup 2 +/, Mn/sup 2 +/, and Mg/sup 2 +/ to the protein. Partial delipidation of the protein was performed by using either the organic solvent extraction procedure or the silicic acid column chromatography. Control experiments indicated that the Ca/sup 2 +/ transport properties of the isolated protein were not due to the contaminating phospholipids. A complete delipidation procedure was developd by using Sephadex LH-20 column chromatography. Further characterization of the physical and chemicalmore » properties of the delipidated protein showed that delipidated protein becomes more hydrophobic in the presence of Ca/sup 2 +/ and alkaline pH in the organic solvent extraction experiments. Two possible models of calciphorin-mediated Ca/sup 2 +/ transport in mitochondria are proposed. (PCS)« less

NMR studies reveal the role of biomembranes in modulating ligand binding and release by intracellular bile acid binding proteins.

PubMed

Pedò, Massimo; Löhr, Frank; D'Onofrio, Mariapina; Assfalg, Michael; Dötsch, Volker; Molinari, Henriette

2009-12-18

Bile acid molecules are transferred vectorially between basolateral and apical membranes of hepatocytes and enterocytes in the context of the enterohepatic circulation, a process regulating whole body lipid homeostasis. This work addresses the role of the cytosolic lipid binding proteins in the intracellular transfer of bile acids between different membrane compartments. We present nuclear magnetic resonance (NMR) data describing the ternary system composed of the bile acid binding protein, bile acids, and membrane mimetic systems, such as anionic liposomes. This work provides evidence that the investigated liver bile acid binding protein undergoes association with the anionic membrane and binding-induced partial unfolding. The addition of the physiological ligand to the protein-liposome mixture is capable of modulating this interaction, shifting the equilibrium towards the free folded holo protein. An ensemble of NMR titration experiments, based on nitrogen-15 protein and ligand observation, confirm that the membrane and the ligand establish competing binding equilibria, modulating the cytoplasmic permeability of bile acids. These results support a mechanism of ligand binding and release controlled by the onset of a bile salt concentration gradient within the polarized cell. The location of a specific protein region interacting with liposomes is highlighted.

Identification of the components of a glycolytic enzyme metabolon on the human red blood cell membrane.

PubMed

Puchulu-Campanella, Estela; Chu, Haiyan; Anstee, David J; Galan, Jacob A; Tao, W Andy; Low, Philip S

2013-01-11

Glycolytic enzymes (GEs) have been shown to exist in multienzyme complexes on the inner surface of the human erythrocyte membrane. Because no protein other than band 3 has been found to interact with GEs, and because several GEs do not bind band 3, we decided to identify the additional membrane proteins that serve as docking sites for GE on the membrane. For this purpose, a method known as "label transfer" that employs a photoactivatable trifunctional cross-linking reagent to deliver a biotin from a derivatized GE to its binding partner on the membrane was used. Mass spectrometry analysis of membrane proteins that were biotinylated following rebinding and photoactivation of labeled GAPDH, aldolase, lactate dehydrogenase, and pyruvate kinase revealed not only the anticipated binding partner, band 3, but also the association of GEs with specific peptides in α- and β-spectrin, ankyrin, actin, p55, and protein 4.2. More importantly, the labeled GEs were also found to transfer biotin to other GEs in the complex, demonstrating for the first time that GEs also associate with each other in their membrane complexes. Surprisingly, a new GE binding site was repeatedly identified near the junction of the membrane-spanning and cytoplasmic domains of band 3, and this binding site was confirmed by direct binding studies. These results not only identify new components of the membrane-associated GE complexes but also provide molecular details on the specific peptides that form the interfacial contacts within each interaction.

Identification of the Components of a Glycolytic Enzyme Metabolon on the Human Red Blood Cell Membrane*

PubMed Central

Puchulu-Campanella, Estela; Chu, Haiyan; Anstee, David J.; Galan, Jacob A.; Tao, W. Andy; Low, Philip S.

2013-01-01

Glycolytic enzymes (GEs) have been shown to exist in multienzyme complexes on the inner surface of the human erythrocyte membrane. Because no protein other than band 3 has been found to interact with GEs, and because several GEs do not bind band 3, we decided to identify the additional membrane proteins that serve as docking sites for GE on the membrane. For this purpose, a method known as “label transfer” that employs a photoactivatable trifunctional cross-linking reagent to deliver a biotin from a derivatized GE to its binding partner on the membrane was used. Mass spectrometry analysis of membrane proteins that were biotinylated following rebinding and photoactivation of labeled GAPDH, aldolase, lactate dehydrogenase, and pyruvate kinase revealed not only the anticipated binding partner, band 3, but also the association of GEs with specific peptides in α- and β-spectrin, ankyrin, actin, p55, and protein 4.2. More importantly, the labeled GEs were also found to transfer biotin to other GEs in the complex, demonstrating for the first time that GEs also associate with each other in their membrane complexes. Surprisingly, a new GE binding site was repeatedly identified near the junction of the membrane-spanning and cytoplasmic domains of band 3, and this binding site was confirmed by direct binding studies. These results not only identify new components of the membrane-associated GE complexes but also provide molecular details on the specific peptides that form the interfacial contacts within each interaction. PMID:23150667

Tunable Membrane Binding of the Intrinsically Disordered Dehydrin Lti30, a Cold-Induced Plant Stress Protein[W

PubMed Central

Eriksson, Sylvia K.; Kutzer, Michael; Procek, Jan; Gröbner, Gerhard; Harryson, Pia

2011-01-01

Dehydrins are intrinsically disordered plant proteins whose expression is upregulated under conditions of desiccation and cold stress. Their molecular function in ensuring plant survival is not yet known, but several studies suggest their involvement in membrane stabilization. The dehydrins are characterized by a broad repertoire of conserved and repetitive sequences, out of which the archetypical K-segment has been implicated in membrane binding. To elucidate the molecular mechanism of these K-segments, we examined the interaction between lipid membranes and a dehydrin with a basic functional sequence composition: Lti30, comprising only K-segments. Our results show that Lti30 interacts electrostatically with vesicles of both zwitterionic (phosphatidyl choline) and negatively charged phospholipids (phosphatidyl glycerol, phosphatidyl serine, and phosphatidic acid) with a stronger binding to membranes with high negative surface potential. The membrane interaction lowers the temperature of the main lipid phase transition, consistent with Lti30’s proposed role in cold tolerance. Moreover, the membrane binding promotes the assembly of lipid vesicles into large and easily distinguishable aggregates. Using these aggregates as binding markers, we identify three factors that regulate the lipid interaction of Lti30 in vitro: (1) a pH dependent His on/off switch, (2) phosphorylation by protein kinase C, and (3) reversal of membrane binding by proteolytic digest. PMID:21665998

Osmoregulated ABC-transport system of Lactococcus lactis senses water stress via changes in the physical state of the membrane.

PubMed

van der Heide, T; Poolman, B

2000-06-20

An osmoregulated ABC transporter (OpuA) with novel structural features has been identified that responds to water stress. This glycine betaine transport system consists of an ATP-binding/hydrolyzing subunit (OpuAA) and a protein (OpuABC) that contains both the translocator and the substrate-binding domain. The components of OpuA have been overexpressed, purified, and functionally incorporated into liposomes with an ATP-regenerating system in the vesicle lumen. A transmembrane osmotic gradient (outside hyperosmotic relative to the inside) of both ionic and nonionic compounds was able to osmotically activate OpuA in the proteoliposomal system. Hypoosmotic medium conditions inhibited the basal activity of the system. The data show that OpuAA and OpuABC are sufficient for osmoregulated transport, indicating that OpuA can act both as osmosensor and osmoregulator. Strikingly, OpuA could also be activated by low concentrations of cationic and anionic amphipaths, which interact with the membrane. This result indicates that activation by a transmembrane osmotic gradient is mediated by changes in membrane properties/protein-lipid interactions.

Osmoregulated ABC-transport system of Lactococcus lactis senses water stress via changes in the physical state of the membrane

PubMed Central

van der Heide, Tiemen; Poolman, Bert

2000-01-01

An osmoregulated ABC transporter (OpuA) with novel structural features has been identified that responds to water stress. This glycine betaine transport system consists of an ATP-binding/hydrolyzing subunit (OpuAA) and a protein (OpuABC) that contains both the translocator and the substrate-binding domain. The components of OpuA have been overexpressed, purified, and functionally incorporated into liposomes with an ATP-regenerating system in the vesicle lumen. A transmembrane osmotic gradient (outside hyperosmotic relative to the inside) of both ionic and nonionic compounds was able to osmotically activate OpuA in the proteoliposomal system. Hypoosmotic medium conditions inhibited the basal activity of the system. The data show that OpuAA and OpuABC are sufficient for osmoregulated transport, indicating that OpuA can act both as osmosensor and osmoregulator. Strikingly, OpuA could also be activated by low concentrations of cationic and anionic amphipaths, which interact with the membrane. This result indicates that activation by a transmembrane osmotic gradient is mediated by changes in membrane properties/protein–lipid interactions. PMID:10860977

Interaction studies reveal specific recognition of an anti-inflammatory polyphosphorhydrazone dendrimer by human monocytes

NASA Astrophysics Data System (ADS)

Ledall, Jérémy; Fruchon, Séverine; Garzoni, Matteo; Pavan, Giovanni M.; Caminade, Anne-Marie; Turrin, Cédric-Olivier; Blanzat, Muriel; Poupot, Rémy

2015-10-01

Dendrimers are nano-materials with perfectly defined structure and size, and multivalency properties that confer substantial advantages for biomedical applications. Previous work has shown that phosphorus-based polyphosphorhydrazone (PPH) dendrimers capped with azabisphosphonate (ABP) end groups have immuno-modulatory and anti-inflammatory properties leading to efficient therapeutic control of inflammatory diseases in animal models. These properties are mainly prompted through activation of monocytes. Here, we disclose new insights into the molecular mechanisms underlying the anti-inflammatory activation of human monocytes by ABP-capped PPH dendrimers. Following an interdisciplinary approach, we have characterized the physicochemical and biological behavior of the lead ABP dendrimer with model and cell membranes, and compared this experimental set of data to predictive computational modelling studies. The behavior of the ABP dendrimer was compared to the one of an isosteric analog dendrimer capped with twelve azabiscarboxylate (ABC) end groups instead of twelve ABP end groups. The ABC dendrimer displayed no biological activity on human monocytes, therefore it was considered as a negative control. In detail, we show that the ABP dendrimer can bind both non-specifically and specifically to the membrane of human monocytes. The specific binding leads to the internalization of the ABP dendrimer by human monocytes. On the contrary, the ABC dendrimer only interacts non-specifically with human monocytes and is not internalized. These data indicate that the bioactive ABP dendrimer is recognized by specific receptor(s) at the surface of human monocytes.Dendrimers are nano-materials with perfectly defined structure and size, and multivalency properties that confer substantial advantages for biomedical applications. Previous work has shown that phosphorus-based polyphosphorhydrazone (PPH) dendrimers capped with azabisphosphonate (ABP) end groups have immuno-modulatory and anti-inflammatory properties leading to efficient therapeutic control of inflammatory diseases in animal models. These properties are mainly prompted through activation of monocytes. Here, we disclose new insights into the molecular mechanisms underlying the anti-inflammatory activation of human monocytes by ABP-capped PPH dendrimers. Following an interdisciplinary approach, we have characterized the physicochemical and biological behavior of the lead ABP dendrimer with model and cell membranes, and compared this experimental set of data to predictive computational modelling studies. The behavior of the ABP dendrimer was compared to the one of an isosteric analog dendrimer capped with twelve azabiscarboxylate (ABC) end groups instead of twelve ABP end groups. The ABC dendrimer displayed no biological activity on human monocytes, therefore it was considered as a negative control. In detail, we show that the ABP dendrimer can bind both non-specifically and specifically to the membrane of human monocytes. The specific binding leads to the internalization of the ABP dendrimer by human monocytes. On the contrary, the ABC dendrimer only interacts non-specifically with human monocytes and is not internalized. These data indicate that the bioactive ABP dendrimer is recognized by specific receptor(s) at the surface of human monocytes. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr03884g

Luteinizing hormone-releasing hormone inactivation by purified pituitary plasma membranes: effects of receptor-binding studies.

PubMed

Clayton, R N; Shakespear, R A; Duncan, J A; Marshall, J C

1979-05-01

Inactivation of LHRH by purified bovine pituitary plasma membranes was studied in vitro. After incubation of [125I]iodo-LHRH with plasma membranes, the amount of tracer bound to the pellet was measured, and the integrity of the unbound tracer in the supernatant was assessed. Reduction in ability to bind to anti-LHRH serum and to rebind to plasma membranes together with altered electrophoretic mobility on polyacrylamide gels showed that the unbound [125I]iodo-LHRH was inactivated. LHRH inactivation occurred rapidly and was dependent upon membrane concentration and incubation temperature. These results indicate that hormone inactivation must be taken into account in the interpretation of LHRH-receptor interactions. During 37 C incubations, the apparent absence of specific LHRH binding can be explained by inactivation of tracer hormone. Significant LHRH inactivation also occurred at 0 C, which in part explains the insensitivity of LHRH receptor assays. Assessment of LHRH inactivation by different particulate subcellular fractions of pituitary tissue showed that the inactivating enzyme was associated with the plasma membranes; other organelles did not alter LHRH. The enzyme appeared to be an integral part of the plasma membrane structure, since enzymic activity could not be removed by washing without reducing specific LHRH binding. Additionally, reduction of LHRH inactivation by the inhibitors Bacitracin and Trasylol and by magnesium was also accompanied by reduced LHRH binding. Previous studies have shown that the majority of LHRH binding to pituitary plasma membranes is to the low affinity site (approximately 10(-6) M), but the significance of this binding has been uncertain. Our findings indicate that low affinity binding probably represents binding of LHRH to the inactivating enzyme. The LHRH analog, D-Ser6(TBu), des Gly10, ethylamide, has greater biological activity than LHRH and is not inactivated to a significant extent by pituitary plasma membranes. The enhanced biological activity of the analog, therefore, may be due to its resistance to inactivation by enzymes on the pituitary cell surface. The membrane-associated inactivating enzyme could play an important role in vivo in determining the concentration of intact LHRH available at the receptor site which initiates gonadotropin release.

Membrane Modulates Affinity for Calcium Ion to Create an Apparent Cooperative Binding Response by Annexin a5

PubMed Central

Gauer, Jacob W.; Knutson, Kristofer J.; Jaworski, Samantha R.; Rice, Anne M.; Rannikko, Anika M.; Lentz, Barry R.; Hinderliter, Anne

2013-01-01

Isothermal titration calorimetry was used to characterize the binding of calcium ion (Ca2+) and phospholipid to the peripheral membrane-binding protein annexin a5. The phospholipid was a binary mixture of a neutral and an acidic phospholipid, specifically phosphatidylcholine and phosphatidylserine in the form of large unilamellar vesicles. To stringently define the mode of binding, a global fit of data collected in the presence and absence of membrane concentrations exceeding protein saturation was performed. A partition function defined the contribution of all heat-evolving or heat-absorbing binding states. We find that annexin a5 binds Ca2+ in solution according to a simple independent-site model (solution-state affinity). In the presence of phosphatidylserine-containing liposomes, binding of Ca2+ differentiates into two classes of sites, both of which have higher affinity compared with the solution-state affinity. As in the solution-state scenario, the sites within each class were described with an independent-site model. Transitioning from a solution state with lower Ca2+ affinity to a membrane-associated, higher Ca2+ affinity state, results in cooperative binding. We discuss how weak membrane association of annexin a5 prior to Ca2+ influx is the basis for the cooperative response of annexin a5 toward Ca2+, and the role of membrane organization in this response. PMID:23746516

Membrane Stored Curvature Elastic Stress Modulates Recruitment of Maintenance Proteins PspA and Vipp1.

PubMed

McDonald, Christopher; Jovanovic, Goran; Ces, Oscar; Buck, Martin

2015-09-01

Phage shock protein A (PspA), which is responsible for maintaining inner membrane integrity under stress in enterobacteria, and vesicle-inducting protein in plastids 1 (Vipp1), which functions for membrane maintenance and thylakoid biogenesis in cyanobacteria and plants, are similar peripheral membrane-binding proteins. Their homologous N-terminal amphipathic helices are required for membrane binding; however, the membrane features recognized and required for expressing their functionalities have remained largely uncharacterized. Rigorously controlled, in vitro methodologies with lipid vesicles and purified proteins were used in this study and provided the first biochemical and biophysical characterizations of membrane binding by PspA and Vipp1. Both proteins are found to sense stored curvature elastic (SCE) stress and anionic lipids within the membrane. PspA has an enhanced sensitivity for SCE stress and a higher affinity for the membrane than Vipp1. These variations in binding may be crucial for some of the proteins' differing roles in vivo. Assays probing the transcriptional regulatory function of PspA in the presence of vesicles showed that a relief of transcription inhibition occurs in an SCE stress-specific manner. This in vitro recapitulation of membrane stress-dependent transcription control suggests that the Psp response may be mounted in vivo when a cell's inner membrane experiences increased SCE stress. All cell types maintain the integrity of their membrane systems. One widely distributed membrane stress response system in bacteria is the phage shock protein (Psp) system. The central component, peripheral membrane protein PspA, which mitigates inner membrane stress in bacteria, has a counterpart, Vipp1, which functions for membrane maintenance and thylakoid biogenesis in plants and photosynthetic bacteria. Membrane association of both these proteins is accepted as playing a pivotal role in their functions. Here we show that direct membrane binding by PspA and Vipp1 is driven by two physio-chemical signals, one of which is membrane stress specific. Our work points to alleviation of membrane stored curvature elastic stress by amphipathic helix insertions as an attractive mechanism for membrane maintenance by PspA and Vipp1. Furthermore, the identification of a physical, stress-related membrane signal suggests a unilateral mechanism that promotes both binding of PspA and induction of the Psp response. Copyright © 2015 McDonald et al.

p-( sup 125 I)iodoclonidine is a partial agonist at the alpha 2-adrenergic receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gerhardt, M.A.; Wade, S.M.; Neubig, R.R.

1990-08-01

The binding properties of p-(125I)iodoclonidine (( 125I)PIC) to human platelet membranes and the functional characteristics of PIC are reported. (125I)PIC bound rapidly and reversibly to platelet membranes, with a first-order association rate constant (kon) at room temperature of 8.0 +/- 2.7 x 10(6) M-1 sec-1 and a dissociation rate constant (koff) of 2.0 +/- 0.8 x 10(-3) sec-1. Scatchard plots of specific (125I)PIC binding (0.1-5 nM) were linear, with a Kd of 1.2 +/- 0.1 nM. (125I)PIC bound to the same number of high affinity sites as the alpha 2-adrenergic receptor (alpha 2-AR) full agonist (3H) bromoxidine (UK14,304), which representedmore » approximately 40% of the sites bound by the antagonist (3H)yohimbine. Guanosine 5'-(beta, gamma-imido)triphosphate greatly reduced the amount of (125I)PIC bound (greater than 80%), without changing the Kd of the residual binding. In competition experiments, the alpha 2-AR-selective ligands yohimbine, bromoxidine, oxymetazoline, clonidine, p-aminoclonidine, (-)-epinephrine, and idazoxan all had Ki values in the low nanomolar range, whereas prazosin, propranolol, and serotonin yielded Ki values in the micromolar range. Epinephrine competition for (125I)PIC binding was stereoselective. Competition for (3H)bromoxidine binding by PIC gave a Ki of 1.0 nM (nH = 1.0), whereas competition for (3H)yohimbine could be resolved into high and low affinity components, with Ki values of 3.7 and 84 nM, respectively. PIC had minimal agonist activity in inhibiting adenylate cyclase in platelet membranes, but it potentiated platelet aggregation induced by ADP with an EC50 of 1.5 microM. PIC also inhibited epinephrine-induced aggregation, with an IC50 of 5.1 microM. Thus, PIC behaves as a partial agonist in a human platelet aggregation assay. (125I)PIC binds to the alpha 2B-AR in NG-10815 cell membranes with a Kd of 0.5 +/- 0.1 nM.« less

Membrane Association of the PTEN Tumor Suppressor: Electrostatic Interaction with Phos-phatidylserine-Containing Bilayers and Regulatory Role of the C-Terminal Tail

PubMed Central

Shenoy, Siddharth S.; Nanda, Hirsh; Lösche, Mathias

2012-01-01

The phosphatidylinositolphosphate phosphatase PTEN is the second most frequently mutated protein in human tumors. Its membrane association, allosteric activation and membrane dissociation are poorly understood. We recently reported PTEN binding affinities to membranes of different compositions and a preliminary investigation of the protein-membrane complex with neutron reflectometry (NR). Here we use NR to validate molecular dynamics (MD) simulations of the protein and study conformational differences of the protein in solution and on anionic membranes. NR shows that full-length PTEN binds to such membranes roughly in the conformation and orientation suggested by the crystal structure of a truncated PTEN protein, in contrast with a recently presented model which suggested that membrane binding depends critically on the SUMOylation of the CBR3 loop of PTEN’s C2 domain. Our MD simulations confirm that PTEN is peripherally bound to the bilayer surface and show slight differences of the protein structure in solution and in the membrane-bound state, where the protein body flattens against the bilayer surface. PTEN’s C2 domain binds phosphatidylserine (PS) tightly through its CBR3 loop, and its phosphatase domain also forms electrostatic interactions with PS. NR and MD results show consistently that PTEN’s unstructured, anionic C-terminal tail is repelled from the bilayer surface. In contrast, this tail is tightly tugged against the C2 domain in solution, partially obstructing the membrane-binding interface of the protein. Arresting the C-terminal tail in this conformation by phosphorylation may provide a control mechanism for PTEN’s membrane binding and activity. PMID:23073177

Membrane association of the PTEN tumor suppressor: electrostatic interaction with phosphatidylserine-containing bilayers and regulatory role of the C-terminal tail.

PubMed

Shenoy, Siddharth S; Nanda, Hirsh; Lösche, Mathias

2012-12-01

The phosphatidylinositolphosphate phosphatase PTEN is the second most frequently mutated protein in human tumors. Its membrane association, allosteric activation and membrane dissociation are poorly understood. We recently reported PTEN binding affinities to membranes of different compositions (Shenoy et al., 2012, PLoS ONE 7, e32591) and a preliminary investigation of the protein-membrane complex with neutron reflectometry (NR). Here we use NR to validate molecular dynamics (MD) simulations of the protein and study conformational differences of the protein in solution and on anionic membranes. NR shows that full-length PTEN binds to such membranes roughly in the conformation and orientation suggested by the crystal structure of a truncated PTEN protein, in contrast with a recently presented model which suggested that membrane binding depends critically on the SUMOylation of the CBR3 loop of PTEN's C2 domain. Our MD simulations confirm that PTEN is peripherally bound to the bilayer surface and show slight differences of the protein structure in solution and in the membrane-bound state, where the protein body flattens against the bilayer surface. PTEN's C2 domain binds phosphatidylserine (PS) tightly through its CBR3 loop, and its phosphatase domain also forms electrostatic interactions with PS. NR and MD results show consistently that PTEN's unstructured, anionic C-terminal tail is repelled from the bilayer surface. In contrast, this tail is tightly tugged against the C2 domain in solution, partially obstructing the membrane-binding interface of the protein. Arresting the C-terminal tail in this conformation by phosphorylation may provide a control mechanism for PTEN's membrane binding and activity. Copyright © 2012 Elsevier Inc. All rights reserved.

Ion-binding properties of a K+ channel selectivity filter in different conformations.

PubMed

Liu, Shian; Focke, Paul J; Matulef, Kimberly; Bian, Xuelin; Moënne-Loccoz, Pierre; Valiyaveetil, Francis I; Lockless, Steve W

2015-12-08

K(+) channels are membrane proteins that selectively conduct K(+) ions across lipid bilayers. Many voltage-gated K(+) (KV) channels contain two gates, one at the bundle crossing on the intracellular side of the membrane and another in the selectivity filter. The gate at the bundle crossing is responsible for channel opening in response to a voltage stimulus, whereas the gate at the selectivity filter is responsible for C-type inactivation. Together, these regions determine when the channel conducts ions. The K(+) channel from Streptomyces lividians (KcsA) undergoes an inactivation process that is functionally similar to KV channels, which has led to its use as a practical system to study inactivation. Crystal structures of KcsA channels with an open intracellular gate revealed a selectivity filter in a constricted conformation similar to the structure observed in closed KcsA containing only Na(+) or low [K(+)]. However, recent work using a semisynthetic channel that is unable to adopt a constricted filter but inactivates like WT channels challenges this idea. In this study, we measured the equilibrium ion-binding properties of channels with conductive, inactivated, and constricted filters using isothermal titration calorimetry (ITC). EPR spectroscopy was used to determine the state of the intracellular gate of the channel, which we found can depend on the presence or absence of a lipid bilayer. Overall, we discovered that K(+) ion binding to channels with an inactivated or conductive selectivity filter is different from K(+) ion binding to channels with a constricted filter, suggesting that the structures of these channels are different.

Structural insights into the methyl donor recognition model of a novel membrane-binding protein UbiG.

PubMed

Zhu, Yuwei; Jiang, Xuguang; Wang, Chongyuan; Liu, Yang; Fan, Xiaojiao; Zhang, Linjuan; Niu, Liwen; Teng, Maikun; Li, Xu

2016-03-15

UbiG is a SAM-dependent O-methyltransferase, catalyzing two O-methyl transfer steps for ubiquinone biosynthesis in Escherichia coli. UbiG possesses a unique sequence insertion between β4 and α10, which is used for membrane lipid interaction. Interestingly, this sequence insertion also covers the methyl donor binding pocket. Thus, the relationship between membrane binding and entrance of the methyl donor of UbiG during the O-methyl transfer process is a question that deserves further exploration. In this study, we reveal that the membrane-binding region of UbiG gates the entrance of methyl donor. When bound with liposome, UbiG displays an enhanced binding ability toward the methyl donor product S-adenosylhomocysteine. We further employ protein engineering strategies to design UbiG mutants by truncating the membrane interacting region or making it more flexible. The ITC results show that the binding affinity of these mutants to SAH increases significantly compared with that of the wild-type UbiG. Moreover, we determine the structure of UbiG∆(165-187) in complex with SAH. Collectively, our results provide a new angle to cognize the relationship between membrane binding and entrance of the methyl donor of UbiG, which is of benefit for better understanding the O-methyl transfer process for ubiquinone biosynthesis.

Cellular membrane enrichment of self-assembling D-peptides for cell surface engineering.

PubMed

Wang, Huaimin; Wang, Youzhi; Han, Aitian; Cai, Yanbin; Xiao, Nannan; Wang, Ling; Ding, Dan; Yang, Zhimou

2014-06-25

We occasionally found that several self-assembling peptides containing D-amino acids would be preferentially enriched in cellular membranes at self-assembled stages while distributed evenly in the cytoplasma of cells at unassembled stages. Self-assembling peptides containing only Lamino acids distributed evenly in cytoplasma of cells at both self-assembled and unassembled stages. The self-assembling peptides containing D-amino acids could therefore be applied for engineering cell surface with peptides. More importantly, by integrating a protein binding peptide (a PDZ domain binding hexapeptide of WRESAI) with the self-assembling peptide containing D-amino acids, protein could also be introduced to the cell surface. This study not only provided a novel approach to engineer cell surface, but also highlighted the unusual properties and potential applications of self-assembling peptides containing D-amino acids in regenerative medicine, drug delivery, and tissue engineering.

Structure and Function of Caltrin (Calcium Transport Inhibitor) Proteins

PubMed Central

Grasso, Ernesto Javier; Coronel, Carlos Enrique

2017-01-01

Caltrin (calcium transport inhibitor) is a family of small and basic proteins of the mammalian seminal plasma which bind to sperm cells during ejaculation and inhibit the extracellular Ca2+ uptake, preventing the premature acrosomal exocytosis and hyperactivation when sperm cells ascend through the female reproductive tract. The binding of caltrin proteins to specific areas of the sperm surface suggests the existence of caltrin receptors, or precise protein-phospholipid arrangements in the sperm membrane, distributed in the regions where Ca2+ influx may take place. However, the molecular mechanisms of recognition and interaction between caltrin and spermatozoa have not been elucidated. Therefore, the aim of this article is to describe in depth the known structural features and functional properties of caltrin proteins, to find out how they may possibly interact with the sperm membranes to control the intracellular signaling that trigger physiological events required for fertilization. PMID:29308010

Selective effect of cell membrane on synaptic neurotransmission

NASA Astrophysics Data System (ADS)

Postila, Pekka A.; Vattulainen, Ilpo; Róg, Tomasz

2016-01-01

Atomistic molecular dynamics simulations were performed with 13 non-peptidic neurotransmitters (NTs) in three different membrane environments. The results provide compelling evidence that NTs are divided into membrane-binding and membrane-nonbinding molecules. NTs adhere to the postsynaptic membrane surface whenever the ligand-binding sites of their synaptic receptors are buried in the lipid bilayer. In contrast, NTs that have extracellular ligand-binding sites do not have a similar tendency to adhere to the membrane surface. This finding is a seemingly simple yet important addition to the paradigm of neurotransmission, essentially dividing it into membrane-independent and membrane-dependent mechanisms. Moreover, the simulations also indicate that the lipid composition especially in terms of charged lipids can affect the membrane partitioning of NTs. The revised paradigm, highlighting the importance of cell membrane and specific lipids for neurotransmission, should to be of interest to neuroscientists, drug industry and the general public alike.

Interaction of the Spo20 membrane-sensor motif with phosphatidic acid and other anionic lipids, and influence of the membrane environment.

PubMed

Horchani, Habib; de Saint-Jean, Maud; Barelli, Hélène; Antonny, Bruno

2014-01-01

The yeast protein Spo20 contains a regulatory amphipathic motif that has been suggested to recognize phosphatidic acid, a lipid involved in signal transduction, lipid metabolism and membrane fusion. We have investigated the interaction of the Spo20 amphipathic motif with lipid membranes using a bioprobe strategy that consists in appending this motif to the end of a long coiled-coil, which can be coupled to a GFP reporter for visualization in cells. The resulting construct is amenable to in vitro and in vivo experiments and allows unbiased comparison between amphipathic helices of different chemistry. In vitro, the Spo20 bioprobe responded to small variations in the amount of phosphatidic acid. However, this response was not specific. The membrane binding of the probe depended on the presence of phosphatidylethanolamine and also integrated the contribution of other anionic lipids, including phosphatidylserine and phosphatidyl-inositol-(4,5)bisphosphate. Inverting the sequence of the Spo20 motif neither affected the ability of the probe to interact with anionic liposomes nor did it modify its cellular localization, making a stereo-specific mode of phosphatidic acid recognition unlikely. Nevertheless, the lipid binding properties and the cellular localization of the Spo20 alpha-helix differed markedly from that of another amphipathic motif, Amphipathic Lipid Packing Sensor (ALPS), suggesting that even in the absence of stereo specific interactions, amphipathic helices can act as subcellular membrane targeting determinants in a cellular context.

Direct observation of the influence of cardiolipin and antibiotics on lipid II binding to MurJ

NASA Astrophysics Data System (ADS)

Bolla, Jani Reddy; Sauer, Joshua B.; Wu, Di; Mehmood, Shahid; Allison, Timothy M.; Robinson, Carol V.

2018-03-01

Translocation of lipid II across the cytoplasmic membrane is essential in peptidoglycan biogenesis. Although most steps are understood, identifying the lipid II flippase has yielded conflicting results, and the lipid II binding properties of two candidate flippases—MurJ and FtsW—remain largely unknown. Here we apply native mass spectrometry to both proteins and characterize lipid II binding. We observed lower levels of lipid II binding to FtsW compared to MurJ, consistent with MurJ having a higher affinity. Site-directed mutagenesis of MurJ suggests that mutations at A29 and D269 attenuate lipid II binding to MurJ, whereas chemical modification of A29 eliminates binding. The antibiotic ramoplanin dissociates lipid II from MurJ, whereas vancomycin binds to form a stable complex with MurJ:lipid II. Furthermore, we reveal cardiolipins associate with MurJ but not FtsW, and exogenous cardiolipins reduce lipid II binding to MurJ. These observations provide insights into determinants of lipid II binding to MurJ and suggest roles for endogenous lipids in regulating substrate binding.

The bile acid-sensitive ion channel (BASIC) is activated by alterations of its membrane environment.

PubMed

Schmidt, Axel; Lenzig, Pia; Oslender-Bujotzek, Adrienne; Kusch, Jana; Lucas, Susana Dias; Gründer, Stefan; Wiemuth, Dominik

2014-01-01

The bile acid-sensitive ion channel (BASIC) is a member of the DEG/ENaC family of ion channels. Channels of this family are characterized by a common structure, their physiological functions and modes of activation, however, are diverse. Rat BASIC is expressed in brain, liver and intestinal tract and activated by bile acids. The physiological function of BASIC and its mechanism of bile acid activation remain a puzzle. Here we addressed the question whether amphiphilic bile acids activate BASIC by directly binding to the channel or indirectly by altering the properties of the surrounding membrane. We show that membrane-active substances other than bile acids also affect the activity of BASIC and that activation by bile acids and other membrane-active substances is non-additive, suggesting that BASIC is sensitive for changes in its membrane environment. Furthermore based on results from chimeras between BASIC and ASIC1a, we show that the extracellular and the transmembrane domains are important for membrane sensitivity.

Quantitation of Membrane-Ligand Interactions Using Backscattering Interferometry

PubMed Central

Baksh, Michael M.; Kussrow, Amanda K.; Mileni, Mauro; Finn, M.G.; Bornhop, Darryl J.

2011-01-01

Though membrane-associated proteins are ubiquitous within all living organisms and represent the majority of drug targets, a general method for direct, label-free measurement of ligand binding to native membranes has not been reported. Here we show backscattering interferometry (BSI) to be a viable technique for quantifying ligand-receptor binding affinities in a variety of membrane environments. By detecting minute changes in the refractive index of a solution, BSI allows binding interactions of proteins with their ligands to be measured at picomolar concentrations. Equilibrium binding constants in the micromolar to picomolar range were obtained for small- and large-molecule interactions in both synthetic- and cell-derived membranes without the use of labels or supporting substrates. The simple and low-cost hardware, high sensitivity, and label-free nature of BSI should make it readily applicable to the study of many membrane-associated proteins of biochemical and pharmacological interest. PMID:21399645

Investigation of Anion-Exchange and Immunoaffinity Particle-Loaded Membranes for the Isolation of Charged Organic Analytes from Water

USGS Publications Warehouse

Dombrowski, T.R.; Wilson, G.S.; Thurman, E.M.

1998-01-01

Anion-exchange and immunoaffinity particle loaded membranes (PLMs) were investigated as a mechanism for the isolation of charged organic analytes from water. Kinetic properties determined theoretically included dynamic capacity, pressure drop (??P), residence and diffusion times (Tr, Td), and total membrane porosity (???T). These properties were confirmed through experimental evaluation, and the PLM method showed significant improvement over conventional solid-phase extraction (SPE) and ion-exchange formats. Recoveries of more than 90% were observed for a variety of test compounds at flow rates up to 70 mL/min (equipment-limited maximum flow rate). A fast-flow immunoaffinity column was developed using antibodies (Abs) attached to the PLMs. Reproducible recoveries (88% ?? 4%) were observed at flow rates up to 70 mL/min for the antibody (Ab)-loaded PLMs. Findings indicate increased selectivity over anion-exchange PLMs and conventional SPE or ion-exchange methods and rapid Ab-antigen binding rates given the excellent mass-transfer characteristics of the PLMs.

Functional characterization of chitin-binding lectin from Solanum integrifolium containing anti-fungal and insecticidal activities.

PubMed

Chen, Chang-Shan; Chen, Chun-Yi; Ravinath, Divya Malathy; Bungahot, Agustina; Cheng, Chi-Ping; You, Ren-In

2018-01-03

Along with the rapid development of glycomic tools, the study of lectin-carbohydrate interactions has expanded, opening the way for applications in the fields of analytic, diagnostic, and drug delivery. Chitin-binding lectins (CBLs) play roles in immune defense against chitin-containing pathogens. CBLs from species of the Solanaceae family, such as tomato, potato and jimsonweed, display different binding specificities to sugar chains containing poly-N-acetyllactosamine. In this report, CBLs from Solanum integrifolium were isolated by ion exchange chromatography. The fractions showed hemagglutination activity (HA). The recombinant CBL in the 293F cell culture supernatant was able to inhibit the growth of Rhizoctonia solani and Colletotrichum gloeosporioide. Furthermore, the carbohydrate-binding property of CBLs was confirmed with the inhibition of HA. Binding of CBL to Spodoptera frugiperda (sf21) insect cells can partly be inhibited by N-Acetylglucosamine (GlcNAc), which is related to decrease mitochondrial membrane potential of sf21 cells. The results showed that CBL exhibited antifungal properties and inhibited insect cell growth, which is directly correlated to the lectin-carbohydrate interaction. Further identification and characterization of CBLs will help to broaden their scope of application in plant defense and in biomedical applications.

Pyrethroid receptor in the insect Na sup + channel: Alteration of its properties in pyrethroid-resistant flies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pauron, D.; Barhanin, J.; Amichot, M.

1989-02-21

Resistance to insecticides is a major problem in agriculture. ({sup 3}H)Saxitoxin binding experiments have shown that pyrethroid-sensitive and pyrethroid-resistant flies have the same amount of Na{sup +} channel protein in their brain membranes. Also, although flies are resistant to pyrethroids, they remain as sensitive to batrachotoxin, which is another type of Na{sup +} channel activators, as pyrethroid-sensitive flies. Pyrethroid binding sites have been characterized by use of the properties of pyrethroids to increase the specific ({sup 3}H)batrachotoxinin A 20{alpha}-benzoate binding component. K{sub 0.5} values for association of pyrethroids at the Na{sup +} channel of pyrethroid-sensitive flies are in the rangemore » of 0.15-0.25 {mu}M. Conversely, pyrethroids do not produce a significant increase of ({sup 3}H)batrachotoxinin A 20{alpha}-benzoate binding in pyrethroid-resistant flies even at high concentrations of the insecticide. It is concluded that linkage between pyrethroid and batrachotoxin binding sites is altered in the pyrethroid-resistant fly strains. This alteration is probably due to a drastically decreased affinity of the Na{sup +} channel for pyrethroids.« less

Expression, subcellular localization and regulation of sigma receptor in retinal Müller cells

PubMed Central

Jiang, Guoliang; Mysona, Barbara; Dun, Ying; Gnana-Prakasam, Jaya P.; Pabla, Navjotsin; Li, Weiguo; Dong, Zheng; Ganapathy, Vadivel; Smith, Sylvia B.

2013-01-01

Purpose Sigma receptors (σR) are non-opioid, non-phencyclidine binding sites with robust neuroprotective properties. σR1 is expressed in brain oligodendrocytes, but its expression and binding capacity have not been analyzed in retinal glial cells. This study examined the expression, subcellular localization, binding activity and regulation of σR1 in retinal Müller cells. Methods Primary mouse Müller cells (1°MC) were analyzed by RT-PCR, immunoblotting and immunocytochemistry for the expression of σR1 and data were compared to the rat Müller cell line, rMC-1 and rat ganglion cell line, RGC-5. Confocal microscopy was used to determine the subcellular σR1 location in 1°MC. Membranes prepared from these cells were used for binding assays using [3H]-pentazocine (PTZ). The kinetics of binding, the ability of various σR1 ligands to compete with σR1 binding and the effects of nitric oxide (NO) and reactive oxygen species (ROS) donors on binding were examined. Results σR1 is expressed in 1°MC and is localized to the nuclear and endoplasmic reticulum membranes. Binding assays showed that in 1°MCs, rMC-1 and RGC-5 cells, the binding of PTZ was saturable. [3H]-PTZ bound with high affinity in RGC-5 and rMC-1 cells and the binding was similarly robust in 1°MC. Competition studies showed marked inhibition of [3H]-PTZ binding in the presence of σR1-specific ligands. Incubation of cells with NO and ROS donors markedly increased σR1 binding activity. Conclusions Müller cells express σR1 and demonstrate robust σR1 binding activity, which is inhibited by σR1 ligands and is stimulated during oxidative stress. The potential of Müller cells to bind σR1 ligands may prove beneficial in retinal degenerative diseases such as diabetic retinopathy. PMID:17122151

Expression, subcellular localization, and regulation of sigma receptor in retinal muller cells.

PubMed

Jiang, Guoliang; Mysona, Barbara; Dun, Ying; Gnana-Prakasam, Jaya P; Pabla, Navjotsin; Li, Weiguo; Dong, Zheng; Ganapathy, Vadivel; Smith, Sylvia B

2006-12-01

Sigma receptors (sigmaRs) are nonopioid, nonphencyclidine binding sites with robust neuroprotective properties. Type 1 sigmaR1 (sigmaR1) is expressed in brain oligodendrocytes, but its expression and binding capacity have not been analyzed in retinal glial cells. This study examined the expression, subcellular localization, binding activity, and regulation of sigmaR1 in retinal Müller cells. Primary mouse Müller cells (MCs) were analyzed by RT-PCR, immunoblotting, and immunocytochemistry for the expression of sigmaR1, and data were compared with those of the rat Müller cell line (rMC-1) and the rat ganglion cell line (RGC-5). Confocal microscopy was used to determine the subcellular sigmaR1 location in primary mouse MCs. Membranes prepared from these cells were used for binding assays with [3H]-pentazocine (PTZ). The kinetics of binding, the ability of various sigmaR1 ligands to compete with sigmaR1 binding, and the effects of donated nitric oxide (NO) and reactive oxygen species (ROS) on binding were examined. sigmaR1 is expressed in primary mouse MCs and is localized to the nuclear and endoplasmic reticulum membranes. Binding assays showed that in primary mouse MCs, rMC-1, and RGC-5, the binding of PTZ was saturable. [3H]-PTZ bound with high affinity in RGC-5 and rMC-1 cells, and the binding was similarly robust in primary mouse MCs. Competition studies showed marked inhibition of [3H]-PTZ binding in the presence of sigmaR1-specific ligands. Incubation of cells with NO and ROS donors markedly increased sigmaR1 binding activity. MCs express sigmaR1 and demonstrate robust sigmaR1 binding activity, which is inhibited by sigmaR1 ligands and is stimulated during oxidative stress. The potential of Müller cells to bind sigmaR1 ligands may prove beneficial in retinal degenerative diseases such as diabetic retinopathy.

Deacetylation of forskolin catalyzed by bovine brain membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Selfe, S.; Storm, D.R.

1985-11-27

Radiolabeled forskolin, 7-(/sup 3/H-acetyl)-forskolin, was synthesized to explore interactions between forskolin and bovine brain membrane preparations. The radiolabeled derivative was chemically characterized, and found to be indistinquishable from unlabeled forskolin in its ability to stimulate bovine brain adenylate cyclase. Preliminary binding data demonstrated that binding of 7-(/sup 3/H-acetyl)-forskolin to membranes was concentration dependent. However, competition binding studies using a constant concentration of 7-(/sup 3/H-acetyl)-forskolin with increasing levels of unlabeled forskolin showed enhanced binding of the labeled derivative. This suggested that 7-(/sup 3/H-acetyl)-forskolin was degraded by membranes and protected by native forskolin. Incubation of forskolin with membranes and analysis of themore » products by thin layer chromatography and mass spectroscopy showed the formation of 7-desacetylforskolin. The deacetylation of forskolin was monitored by quantitating the release of (/sup 3/H)acetate from 7-(/sup 3/H-acetyl)-forskolin. The reaction was linear with time and protein concentration. These data illustrate that forskolin can be degraded by membranes and indicate that ligand binding studies using labeled forskolin and membrane preparations should be cautiously interpreted.« less

The action of stress hormones on the structure and function of erythrocyte membrane.

PubMed

Mokrushnikov, Pavel V; Panin, Lev E; Zaitsev, Boris N

2015-07-01

The action of a mixture of hormones (cortisol and adrenaline) on erythrocyte membrane during their binding was investigated. Changes in the membrane structure were elucidated by atomic force microscopy; microviscosity of the lipid bilayer and changes in the activity of Na(+),K(+)-ATPase at different concentrations of the hormones in erythrocyte suspension were estimated by the fluorescence method. Cortisol and adrenaline were shown to compete for the binding sites. A hormone that managed to bind nonspecifically to the membrane hindered the binding of another hormone. In a mixture of these hormones, cortisol won a competition for the binding sites; therewith, microviscosity of the membranes increased by 25%, which corresponds to a change in microviscosity produced by the action of cortisol alone. The competitive relationships affected also the Na(+),K(+)-ATPase activity, which was indicated by appearance of the second maximum of enzyme activity. It is assumed that an increase in microviscosity of erythrocyte membrane first raises the Na(+),K(+)-ATPase activity due to a growth of the maximum energy of membrane phonons, and then decreases the activity due to hindering of conformational transitions in the enzyme molecule.

LH-RH binding to purified pituitary plasma membranes: absence of adenylate cyclase activation.

PubMed

Clayton, R N; Shakespear, R A; Marshall, J C

1978-06-01

Purified bovine pituitary plasma membranes possess two specific LH-RH binding sites. The high affinity site (2.5 X 10(9) l/mol) has low capacity (9 X 10(-15) mol/mg membrane protein) while the low affinity site 6.1 X 10(5) l/mol) has a much higher capacity (1.1 X 10(-10) mol/mg). Specific LH-RH binding to plasma membranes is increased 8.5-fold during purification from homogenate whilst adenylate cyclase activity is enriched 7--8-fold. Distribution of specific LH-RH binding to sucrose density gradient interface fractions parallels that of adenylate cyclase activity. Mg2+ and Ca2+ inhibit specific [125I]LH-RH binding at micromolar concentrations. Synthetic LH-RH, up to 250 microgram/ml, failed to stimulate adenylase cyclase activity of the purified bovine membranes. Using a crude 10,800 g rat pituitary membrane preparation, LH-RH similarly failed to activate adenylate cyclase even in the presence of guanyl nucleotides. These data confirm the presence of LH-RH receptor sites on pituitary plasma membranes and suggest that LH-RH-induced gonadotrophin release may be mediated by mechanisms other than activation of adenylate cyclase.

PtdInsP2 and PtdSer cooperate to trap synaptotagmin-1 to the plasma membrane in the presence of calcium

PubMed Central

Pérez-Lara, Ángel; Thapa, Anusa; Nyenhuis, Sarah B; Nyenhuis, David A; Halder, Partho; Tietzel, Michael; Tittmann, Kai; Cafiso, David S; Jahn, Reinhard

2016-01-01

The Ca2+-sensor synaptotagmin-1 that triggers neuronal exocytosis binds to negatively charged membrane lipids (mainly phosphatidylserine (PtdSer) and phosphoinositides (PtdIns)) but the molecular details of this process are not fully understood. Using quantitative thermodynamic, kinetic and structural methods, we show that synaptotagmin-1 (from Rattus norvegicus and expressed in Escherichia coli) binds to PtdIns(4,5)P2 via a polybasic lysine patch in the C2B domain, which may promote the priming or docking of synaptic vesicles. Ca2+ neutralizes the negative charges of the Ca2+-binding sites, resulting in the penetration of synaptotagmin-1 into the membrane, via binding of PtdSer, and an increase in the affinity of the polybasic lysine patch to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2). These Ca2+-induced events decrease the dissociation rate of synaptotagmin-1 membrane binding while the association rate remains unchanged. We conclude that both membrane penetration and the increased residence time of synaptotagmin-1 at the plasma membrane are crucial for triggering exocytotic membrane fusion. DOI: http://dx.doi.org/10.7554/eLife.15886.001 PMID:27791979

Pressure reversal of the action of octanol on postsynaptic membranes from Torpedo.

PubMed Central

Braswell, L. M.; Miller, K. W.; Sauter, J. F.

1984-01-01

Octanol increases the binding of [3H]-acetylcholine to the desensitized state of the nicotinic receptor in postsynaptic membranes prepared from Torpedo californica. This increase in binding results from an increase in the affinity of [3H]-acetylcholine for its receptor without any change in the number of sites or the shape of the acetylcholine binding curve. High pressures of helium (300 atm) decrease [3H]-acetylcholine binding by a mechanism that changes only the affinity of acetylcholine binding. Helium pressure reverses the effect of octanol on the affinity of [3H]-acetylcholine for its receptor. This pressure reversal of the action of octanol at a postsynaptic membrane is consistent either with pressure counteracting an octanol-induced membrane expansion or with independent mechanisms for the actions of octanol and pressure. The data do not conform with a mechanism in which pressure displaces octanol from a binding site on the receptor protein. PMID:6487895

Atrial natriuretic factor receptor heterogeneity in rat tissues

DOE Office of Scientific and Technical Information (OSTI.GOV)

Andresen, J.W.; Kuno, T.; Kamisaki, Y.

1986-03-01

Rat /sup 125/I-atrial natriuretic factor (ANF, 8-33) was used to identify ANF receptors in membrane preparations from rat adrenal gland and lung. When solubilized with Lubrol-PX, the receptors retained a binding profile and properties that correspond to the high affinity and specificity found in crude membranes. Single peaks of binding activity were observed in gel permeation HPLC and density gradient centrifugation analysis of the solubilized preparations. However, when membranes and solubilized preparations were labeled with /sup 125/I-ANF, treated with crosslinking reagent (disuccinimidyl suberate), and analyzed by SDS gel electrophoresis several specifically labeled bands (120,000, 70,000, and 60,000 daltons) were identifiedmore » by autoradiography. The relative distribution of the specifically labeled proteins varied significantly between rat adrenal gland and lung. In adrenal glands the 120K dalton band was the most prominent specifically labeled protein, while the 60K and 70K dalton proteins were labeled to a lesser degree. In lung membranes the lower molecular weight proteins were more prominent. These results suggest the presence of multiple ANF receptor subtypes, the distribution of which varies among tissues. Chromatographic separation and further characterization of these receptors are currently in progress, and preliminary purification studies support this hypothesis.« less

Binding and Fusion of Extracellular Vesicles to the Plasma Membrane of Their Cell Targets.

PubMed

Prada, Ilaria; Meldolesi, Jacopo

2016-08-09

Exosomes and ectosomes, extracellular vesicles of two types generated by all cells at multivesicular bodies and the plasma membrane, respectively, play critical roles in physiology and pathology. A key mechanism of their function, analogous for both types of vesicles, is the fusion of their membrane to the plasma membrane of specific target cells, followed by discharge to the cytoplasm of their luminal cargo containing proteins, RNAs, and DNA. Here we summarize the present knowledge about the interactions, binding and fusions of vesicles with the cell plasma membrane. The sequence initiates with dynamic interactions, during which vesicles roll over the plasma membrane, followed by the binding of specific membrane proteins to their cell receptors. Membrane binding is then converted rapidly into fusion by mechanisms analogous to those of retroviruses. Specifically, proteins of the extracellular vesicle membranes are structurally rearranged, and their hydrophobic sequences insert into the target cell plasma membrane which undergoes lipid reorganization, protein restructuring and membrane dimpling. Single fusions are not the only process of vesicle/cell interactions. Upon intracellular reassembly of their luminal cargoes, vesicles can be regenerated, released and fused horizontally to other target cells. Fusions of extracellular vesicles are relevant also for specific therapy processes, now intensely investigated.

Simple method provides resolution of albumin, lipoprotein, free fraction, and chylomicron to enhance the utility of protein binding assays.

PubMed

Brockman, Adam H; Oller, Haley R; Moreau, Benoît; Kriksciukaite, Kristina; Bilodeau, Mark T

2015-02-12

Medicinal chemists have been encouraged in recent years to embrace high speed protein binding assays. These methods employ dialysis membranes in 96-well format or spin filters. Membrane-based methods do not separate lipoprotein binding from albumin binding and introduce interference despite membrane binding controls. Ultracentrifugation methods, in contrast, do not introduce interference if density gradients can be avoided and they resolve lipoprotein from albumin. A new generation of compact, fast ultracentrifuges facilitates the rapid and fully informative separation of plasma into albumin, albumin/fatty acid complex, lipoprotein, protein-free, and chylomicron fractions with no need of salt or sugar density gradients. We present a simple and fast ultracentrifuge method here for two platinum compounds and a taxane that otherwise bound irreversibly to dialysis membranes and which exhibited distinctive lipoprotein binding behaviors. This new generation of ultracentrifugation methods underscores a need to further discuss protein binding assessments as they relate to medicinal chemistry efforts.

Report of a Workshop on Membrane Biophysics Held June 25 - July 15, 1984.

DTIC Science & Technology

1984-07-15

J.A. (1984) Identification of aggregated Markovian models: application to the nicotinic acetylcholine receptor. Prof. of the Neyman-Kiefer Memorial...for future biological applications of the system. We have also discovered that the kinetic and equilibrium 0 properties of the binding depend...consists of aggregation of phycobiliproteins, -- namely phycoerythrin, phycocyanin , and allophycocyanin, which collectively form the phycobilisomes. Each

Pressure Modulation of the Enzymatic Activity of Phospholipase A2, A Putative Membrane-Associated Pressure Sensor.

PubMed

Suladze, Saba; Cinar, Suleyman; Sperlich, Benjamin; Winter, Roland

2015-10-07

Phospholipases A2 (PLA2) catalyze the hydrolysis reaction of sn-2 fatty acids of membrane phospholipids and are also involved in receptor signaling and transcriptional pathways. Here, we used pressure modulation of the PLA2 activity and of the membrane's physical-chemical properties to reveal new mechanistic information about the membrane association and subsequent enzymatic reaction of PLA2. Although the effect of high hydrostatic pressure (HHP) on aqueous soluble and integral membrane proteins has been investigated to some extent, its effect on enzymatic reactions operating at the water/lipid interface has not been explored, yet. This study focuses on the effect of HHP on the structure, membrane binding and enzymatic activity of membrane-associated bee venom PLA2, covering a pressure range up to 2 kbar. To this end, high-pressure Fourier-transform infrared and high-pressure stopped-flow fluorescence spectroscopies were applied. The results show that PLA2 binding to model biomembranes is not significantly affected by pressure and occurs in at least two kinetically distinct steps. Followed by fast initial membrane association, structural reorganization of α-helical segments of PLA2 takes place at the lipid water interface. FRET-based activity measurements reveal that pressure has a marked inhibitory effect on the lipid hydrolysis rate, which decreases by 75% upon compression up to 2 kbar. Lipid hydrolysis under extreme environmental conditions, such as those encountered in the deep sea where pressures up to the kbar-level are encountered, is hence markedly affected by HHP, rendering PLA2, next to being a primary osmosensor, a good candidate for a sensitive pressure sensor in vivo.

Gating modifier toxins isolated from spider venom: modulation of voltage-gated sodium channels and the role of lipid membranes.

PubMed

Agwa, Akello J; Peigneur, Steve; Chow, Chun Yuen; Lawrence, Nicole; Craik, David J; Tytgat, Jan; King, Glenn F; Henriques, Sonia Troeira; Schroeder, Christina I

2018-04-27

Gating modifier toxins (GMTs) are venom-derived peptides isolated from spiders and other venomous creatures that modulate activity of disease-relevant voltage-gated ion channels and are therefore being pursued as therapeutic leads. The amphipathic surface profile of GMTs has prompted the proposal that some GMTs simultaneously bind to the cell membrane and voltage-gated ion channels in a trimolecular complex. Here we examined whether there is a relationship among spider GMT amphipathicity, membrane binding and potency or selectivity for voltage-gated sodium (NaV) channels. We used NMR spectroscopy and in silico calculations to examine the structures and physicochemical properties of a panel of nine GMTs and deployed surface plasmon resonance to measure GMT affinity for lipids putatively found in proximity to NaV channels. Electrophysiology was used to quantify GMT activity on NaV1.7, an ion channel linked to chronic pain. Selectivity of the peptides was further examined against a panel of NaV channel subtypes. We show that GMTs adsorb to the outer leaflet of anionic lipid bilayers through electrostatic interactions. We did not observe a direct correlation between GMT amphipathicity and affinity for lipid bilayers. Furthermore, GMT-lipid bilayer interactions did not correlate with potency or selectivity for NaVs. We therefore propose that increased membrane binding is unlikely to improve subtype selectivity and that the conserved amphipathic GMT surface profile is an adaptation that facilitates simultaneous modulation of multiple NaVs. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization of kappa 1 and kappa 2 opioid binding sites in frog (Rana esculenta) brain membrane preparation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Benyhe, S.; Varga, E.; Hepp, J.

1990-09-01

The distribution and properties of frog brain kappa-opioid receptor subtypes differ not only from those of the guinea pig brain, but also from that of the rat brain. In guinea pig cerebellum the kappa 1 is the dominant receptor subtype, frog brain contains mainly the kappa 2 subtype, and the distribution of the rat brain subtypes is intermediate between the two others. In competition experiments it has been established that ethylketocyclazocine and N-cyclopropylmethyl-norazidomorphine, which are nonselective kappa-ligands, have relatively high affinities to frog brain membranes. The kappa 2 ligands (Met5)enkephalin-Arg6-Phe7 and etorphine also show high affinities to the frog brain.more » Kappa 1 binding sites measured in the presence of 5 microM/D-Ala2-Leu5/enkephalin represent 25-30% of (3H)ethylketocyclazocine binding in frog brain membranes. The kappa 2 subtype in frog brain resembles more to the mu subtype than the delta subtype of opioid receptors, but it differs from the mu subtype in displaying low affinity toward beta-endorphin and /D-Ala2-(Me)Phe4-Gly5-ol/enkephalin (DAGO). From our data it is evident that the opioid receptor subtypes are already present in the amphibian brain but the differences among them are less pronounced than in mammalian brain.« less

Enzymatic properties and substrate specificity of a bacterial phosphatidylcholine synthase.

PubMed

Aktas, Meriyem; Köster, Stefan; Kizilirmak, Sarah; Casanova, Javier C; Betz, Heidi; Fritz, Christiane; Moser, Roman; Yildiz, Özkan; Narberhaus, Franz

2014-08-01

Phosphatidylcholine (PC) is a rare membrane lipid in bacteria, but is crucial for virulence of the plant pathogen Agrobacterium tumefaciens and various other pathogens. Agrobacterium tumefaciens uses two independent PC biosynthesis pathways. One is dependent on the integral membrane protein PC synthase (Pcs), which catalyzes the conversion of cytidine diphosphate-diacylglycerol (CDP-DAG) and choline to PC, thereby releasing a cytidine monophosphate (CMP). Here, we show that Pcs consists of eight transmembrane segments with its N- and C-termini located in the cytoplasm. A cytoplasmic loop between the second and third membrane helix contains the majority of the conserved amino acids of a CDP-alcohol phosphotransferase motif (DGX2 ARX12 GX3 DX3 D). Using point mutagenesis, we provide evidence for a crucial role of this motif in choline binding and enzyme activity. To study the catalytic features of the enzyme, we established a purification protocol for recombinant Pcs. The enzyme forms stable oligomers and exhibits broad substrate specificity towards choline derivatives. The presence of CDP-DAG and manganese is a prerequisite for cooperative binding of choline. PC formation by Pcs is reversible and proceeds via two successive reactions. In a first choline- and manganese-independent reaction, CDP-DAG is hydrolyzed releasing a CMP molecule. The resulting phosphatidyl intermediate reacts with choline in a second manganese-dependent step to form PC. Pcs and Pcs bind by molecular sieving (1, 2, 3). © 2014 FEBS.

LACTB is a filament-forming protein localized in mitochondria

PubMed Central

Polianskyte, Zydrune; Peitsaro, Nina; Dapkunas, Arvydas; Liobikas, Julius; Soliymani, Rabah; Lalowski, Maciej; Speer, Oliver; Seitsonen, Jani; Butcher, Sarah; Cereghetti, Grazia M.; Linder, Matts D.; Merckel, Michael; Thompson, James; Eriksson, Ove

2009-01-01

LACTB is a mammalian active-site serine protein that has evolved from a bacterial penicillin-binding protein. Penicillin-binding proteins are involved in the metabolism of peptidoglycan, the major bacterial cell wall constituent, implying that LACTB has been endowed with novel biochemical properties during eukaryote evolution. Here we demonstrate that LACTB is localized in the mitochondrial intermembrane space, where it is polymerized into stable filaments with a length extending more than a hundred nanometers. We infer that LACTB, through polymerization, promotes intramitochondrial membrane organization and micro-compartmentalization. These findings have implications for our understanding of mitochondrial evolution and function. PMID:19858488

Characterization of ( sup 3 H)alprazolam binding to central benzodiazepine receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

McCabe, R.T.; Mahan, D.R.; Smith, R.B.

1990-10-01

The binding of the triazolobenzodiazepine ({sup 3}H)alprazolam was studied to characterize the in vitro interactions with benzodiazepine receptors in membrane preparations of rat brain. Studies using nonequilibrium and equilibrium binding conditions for ({sup 3}H)alprazolam resulted in high specific to nonspecific (signal to noise) binding ratios. The binding of ({sup 3}H)alprazolam was saturable and specific with a low nanomolar affinity for benzodiazepine receptors in the rat brain. The Kd was 4.6 nM and the Bmax was 2.6 pmol/mg protein. GABA enhanced ({sup 3}H)alprazolam binding while several benzodiazepine receptor ligands were competitive inhibitors of this drug. Compounds that bind to other receptormore » sites had a very weak or negligible effect on ({sup 3}H)alprazolam binding. Alprazolam, an agent used as an anxiolytic and in the treatment of depression, acts in vitro as a selective and specific ligand for benzodiazepine receptors in the rat brain. The biochemical binding profile does not appear to account for the unique therapeutic properties which distinguish this compound from the other benzodiazepines in its class.« less

Specificity of the weak binding between the phage SPO1 transcription-inhibitory protein, TF1, and SPO1 DNA.

PubMed

Johnson, G G; Geiduschek, E P

1977-04-05

The interaction of the phage SPO1 protein transcription factor 1 (TF1), with DNA has been analyzed by membrane filter binding and by sedimentation methods. Substantially specific binding of TF1 to helical SPO1 DNA can be demonstrated by nitrocellulose filter-binding assays at relatively low ionic strength (0.08). However, TF1-DNA complexes dissociate and reequilibrate relatively rapidly and this makes filter-binding assays unsuitable for quantitative measurements of binding equilibra. Accordingly, the sedimentation properties of TF1-DNA complexes have been explored and a short-column centrifugation assay has been elaborated for quantitative measurements. Preferential binding of TF1 to the hydroxymethyluracil-containing SPO1 DNA has also been demonstrated by short-column centrifugation. TF1 binds relatively weakly and somewhat cooperatively to SPO1 DNA at many sites; TF1-DNA complexes dissociate and reequilibrate rapidly. At 20 degrees C in 0.01 M phosphate, pH 7.5, 0.15 KC1, one molecule of TF1 can bind to approximately every 60 nucleotide pairs of SPO1 DNA.

Binding of /sup 18/F by cell membranes and cell walls of Streptococcus mutans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yotis, W.W.; Zeb, M.; McNulty, J.

1983-07-01

The binding of /sup 18/F to isolated cell membranes and cell walls of Streptococcus mutans GS-5 or other bacteria was assayed. The attachment of /sup 18/F to these cell envelopes proceeded slowly and reached equilibrium within 60 min. /sup 18/F binding was stimulated by Ca/sup 2 +/ (1 mM). The binding of /sup 18/F to cellular components was dependent upon the pH, as well as the amount of /sup 18/F and dose of the binder employed. The binding of /sup 18/F by cell walls prepared from fluoride-sensitive and fluoride-resistant cells of S. salivarius and S. mutans did not differ significantly.more » The pretreatment of cell walls or cell membranes for 60 min at 30 degrees C with 1 mg of RNase, DNase, or trypsin per ml did not influence the binding of /sup 18/F by the walls and membranes of S. mutans GS-5. However, prior exposure of cell membranes to sodium dodecyl sulfate caused a significant reduction in the number of /sup 18/F atoms bound by the membranes. In saturated assay systems, cell membranes of S. mutans GS-5 bound 10(15) to 10(16) atoms of /sup 18/F per mg (dry weight), whereas cell walls from S. mutans GS-5, FA-1, and HS-6 or Actinomyces viscosus T14V and T14AV bound 10(12) to 10(13) atoms of /sup 18/F per mg (dry weight). /sup 18/F in this quantity (10(12) to 10(13) atoms) cannot be detected with the fluoride electrode. The data provide, for the first time, a demonstration of /sup 18/F binding by cell membranes and walls of oral flora.« less

Allostery in a disordered protein: Oxidative modifications to α-Synuclein act distally to regulate membrane binding

PubMed Central

Sevcsik, Eva; Trexler, Adam J.; Dunn, Joanna M.; Rhoades, Elizabeth

2011-01-01

Both oxidative stress and aggregation of the protein α-synuclein (aS) have been implicated as key factors in the etiology of Parkinson’s disease. Specifically, oxidative modifications to aS disrupt its binding to lipid membranes, an interaction considered critical to its native function. Here we seek to provide a mechanistic explanation for this phenomenon by investigating the effects of oxidative nitration of tyrosine residues on the structure of aS and its interaction with lipid membranes. Membrane binding is mediated by the first ~95 residues of aS. We find that nitration of the single tyrosine (Y39) in this domain disrupts binding due to electrostatic repulsion. Moreover, we observe that nitration of the three tyrosines (Y125/133/136) in the C-terminal domain is equally effective in perturbing binding, an intriguing result given that the C-terminus is not thought to interact directly with membranes. Our investigations show that tyrosine nitration results in a change of the conformational states populated by aS in solution, with the most prominent changes occurring in the C-terminal region. These results lead us to suggest that nitration of Y125/133/136 reduces the membrane binding affinity of aS through allosteric coupling by altering the ensemble of conformational states and depopulating those capable of membrane binding. While allostery is a well-established concept for structured proteins, it has only recently been discussed in the context of disordered proteins. We propose that allosteric regulation through modification of specific residues in, or ligand binding to, the C-terminus may even be a general mechanism for modulating aS function. PMID:21491910

The Tip of the Four N-Terminal α-Helices of Clostridium sordellii Lethal Toxin Contains the Interaction Site with Membrane Phosphatidylserine Facilitating Small GTPases Glucosylation

PubMed Central

Varela Chavez, Carolina; Haustant, Georges Michel; Baron, Bruno; England, Patrick; Chenal, Alexandre; Pauillac, Serge; Blondel, Arnaud; Popoff, Michel-Robert

2016-01-01

Clostridium sordellii lethal toxin (TcsL) is a powerful virulence factor responsible for severe toxic shock in man and animals. TcsL belongs to the large clostridial glucosylating toxin (LCGT) family which inactivates small GTPases by glucosylation with uridine-diphosphate (UDP)-glucose as a cofactor. Notably, TcsL modifies Rac and Ras GTPases, leading to drastic alteration of the actin cytoskeleton and cell viability. TcsL enters cells via receptor-mediated endocytosis and delivers the N-terminal glucosylating domain (TcsL-cat) into the cytosol. TcsL-cat was found to preferentially bind to phosphatidylserine (PS)-containing membranes and to increase the glucosylation of Rac anchored to the lipid membrane. We have previously reported that the N-terminal four helical bundle structure (1–93 domain) recognizes a broad range of lipids, but that TcsL-cat specifically binds to PS and phosphatidic acid. Here, we show using mutagenesis that the PS binding site is localized on the tip of the four-helix bundle which is rich in positively-charged amino acids. Residues Y14, V15, F17, and R18 on loop 1, between helices 1 and 2, in coordination with R68 from loop 3, between helices 3 and 4, form a pocket which accommodates L-serine. The functional PS-binding site is required for TcsL-cat binding to the plasma membrane and subsequent cytotoxicity. TcsL-cat binding to PS facilitates a high enzymatic activity towards membrane-anchored Ras by about three orders of magnitude as compared to Ras in solution. The PS-binding site is conserved in LCGTs, which likely retain a common mechanism of binding to the membrane for their full activity towards membrane-bound GTPases. PMID:27023605

Ion channels in artificial bolaamphiphile membranes deposited on sensor chips: optical detection in an ion-channel-based biosensor

NASA Astrophysics Data System (ADS)

Schalkhammer, Thomas G. M.; Weiss-Wichert, Christof; Smetazko, Michaela M.; Valina-Saba, Miriam

1997-06-01

Signal amplification using labels should be replaced by a technique monitoring the biochemical binding event directly. The use of a ligand coupled to an artificial gated membrane ion channel is a new promising strategy. Binding of protein- or DNA/RNA-analytes at ligand modified peptide channels results in an on/off-response of the channel current due to channel closure or distortion. The sensor consists of stable transmembrane channels with a ligand bound covalently at the peptide channel entrance, a sensor chip with a photostructurized hydrophobic polymer frame, a hydrophilic ion conducting membrane support, a lipid membrane incorporating the engineered ion channels, and a current amplifier or a sensitive fluorescence monitor. Detection of channel opening or closure can ether be obtained by directly monitoring membrane conductivity or a transient change of pH or ion concentration within the membrane compartment. This change can be induced by electrochemical or optical means and its decay is directly correlated to the permeability of the membrane. The ion concentration in the sub membrane compartment was monitored by incorporation of fluorescent indicator dyes. To obtain the stable sensor membrane the lipid layer had to be attached on a support and the floating of the second lipid membrane on top of the first one had to be prevented. Both problems do not occur using our new circular C44-C76 bolaamphiphilic lipids consisting of a long hydrophobic core region and two hydrophilic heads. Use of maleic ester-head groups enabled us to easily modify the lipids with amines, thioles, alcohols, phosphates, boronic acid as well as fluorescent dyes. The properties of these membranes were studied using LB and fluorescence techniques. Based on this detection principle miniaturized sensor chips with significantly enhanced sensitivity and large multi analyte arrays are under construction.

Membrane interaction of the N-terminal domain of chemokine receptor CXCR1.

PubMed

Haldar, Sourav; Raghuraman, H; Namani, Trishool; Rajarathnam, Krishna; Chattopadhyay, Amitabha

2010-06-01

The N-terminal domain of chemokine receptors constitutes one of the two critical ligand binding sites, and plays important roles by mediating binding affinity, receptor selectivity, and regulating function. In this work, we monitored the organization and dynamics of a 34-mer peptide of the CXC chemokine receptor 1 (CXCR1) N-terminal domain and its interaction with membranes by utilizing a combination of fluorescence-based approaches and surface pressure measurements. Our results show that the CXCR1 N-domain 34-mer peptide binds vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and upon binding, the tryptophan residues of the peptide experience motional restriction and exhibit red edge excitation shift (REES) of 19nm. These results are further supported by increase in fluorescence anisotropy and mean fluorescence lifetime upon membrane binding. These results constitute one of the first reports demonstrating membrane interaction of the N-terminal domain of CXCR1 and gain relevance in the context of the emerging role of cellular membranes in chemokine signaling.

MFP1 is a thylakoid-associated, nucleoid-binding protein with a coiled-coil structure

PubMed Central

Jeong, Sun Yong; Rose, Annkatrin; Meier, Iris

2003-01-01

Plastid DNA, like bacterial and mitochondrial DNA, is organized into protein–DNA complexes called nucleoids. Plastid nucleoids are believed to be associated with the inner envelope in developing plastids and the thylakoid membranes in mature chloroplasts, but the mechanism for this re-localization is unknown. Here, we present the further characterization of the coiled-coil DNA-binding protein MFP1 as a protein associated with nucleoids and with the thylakoid membranes in mature chloroplasts. MFP1 is located in plastids in both suspension culture cells and leaves and is attached to the thylakoid membranes with its C-terminal DNA-binding domain oriented towards the stroma. It has a major DNA-binding activity in mature Arabidopsis chloroplasts and binds to all tested chloroplast DNA fragments without detectable sequence specificity. Its expression is tightly correlated with the accumulation of thylakoid membranes. Importantly, it is associated in vivo with nucleoids, suggesting a function for MFP1 at the interface between chloroplast nucleoids and the developing thylakoid membrane system. PMID:12930969

Lipid vesicle-mediated affinity chromatography using magnetic activated cell sorting (LIMACS): a novel method to analyze protein-lipid interaction.

PubMed

Bieberich, Erhard

2011-04-26

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids. To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Receptor-mediated internalization of [3H]-neurotensin in synaptosomal preparations from rat neostriatum.

PubMed

Nguyen, Ha Minh Ky; Cahill, Catherine M; McPherson, Peter S; Beaudet, Alain

2002-06-01

Following its binding to somatodendritic receptors, the neuropeptide neurotensin (NT) internalizes via a clathrin-mediated process. In the present study, we investigated whether NT also internalizes presynaptically using synaptosomes from rat neostriatum, a region in which NT1 receptors are virtually all presynaptic. Binding of [(3)H]-NT to striatal synaptosomes in the presence of levocabastine to block NT2 receptors is specific, saturable, and has NT1 binding properties. A significant fraction of the bound radioactivity is resistant to hypertonic acid wash indicating that it is internalized. Internalization of [(3)H]-NT, like that of [(125)I]-transferrin, is blocked by sucrose and low temperature, consistent with endocytosis occurring via a clathrin-dependent pathway. However, contrary to what was reported at the somatodendritic level, neither [(3)H]-NT nor [(125)I]-transferrin internalization in synaptosomes is sensitive to the endocytosis inhibitor phenylarsine oxide. Moreover, treatment of synaptosomes with monensin, which prevents internalized receptors from recycling to the plasma membrane, reduces [(3)H]-NT binding and internalization, suggesting that presynaptic NT1 receptors, in contrast to somatodendritic ones, are recycled back to the plasma membrane. Taken together, these results suggest that NT internalizes in nerve terminals via an endocytic pathway that is related to, but is mechanistically distinct from that responsible for NT internalization in nerve cell bodies.

Phosphorylcholine Allows for Evasion of Bactericidal Antibody by Haemophilus influenzae

PubMed Central

Clark, Sarah E.; Snow, Julian; Li, Jianjun; Zola, Tracey A.; Weiser, Jeffrey N.

2012-01-01

The human pathogen Haemophilus influenzae has the ability to quickly adapt to different host environments through phase variation of multiple structures on its lipooligosaccharide (LPS), including phosphorylcholine (ChoP). During colonization with H. influenzae, there is a selection for ChoP+ phase variants. In a murine model of nasopharyngeal colonization, this selection is lost in the absence of adaptive immunity. Based on previous data highlighting the importance of natural antibody in limiting H. influenzae colonization, the effect of ChoP expression on antibody binding and its bactericidal activity was investigated. Flow cytometric analysis revealed that ChoP+ phase variants had decreased binding of antibody to LPS epitopes compared to ChoP− phase variants. This difference in antibody binding correlated with increased survival of ChoP+ phase variants in the presence of antibody-dependent, complement-mediated killing. ChoP+ phase variants were also more resistant to trypsin digestion, suggesting a general effect on the physical properties of the outer membrane. Moreover, ChoP-mediated protection against antibody binding correlated with increased resilience of outer membrane integrity. Collectively, these data suggest that ChoP expression provides a selective advantage during colonization through ChoP-mediated effects on the accessibility of bactericidal antibody to the cell surface. PMID:22396641

Anchoring antibodies to membranes using a diphtheria toxin T domain-ZZ fusion protein as a pH sensitive membrane anchor.

PubMed

Nizard, P; Liger, D; Gaillard, C; Gillet, D

1998-08-14

We have constructed a fusion protein, T-ZZ, in which the IgG-Fc binding protein ZZ was fused to the C-terminus of the diphtheria toxin transmembrane domain (T domain). While soluble at neutral pH, T-ZZ retained the capacity of the T domain to bind to phospholipid membranes at acidic pH. Once anchored to the membrane, the ZZ part of the protein was capable of binding mouse monoclonal or rabbit polyclonal IgG. Our results show that the T-ZZ protein can function as a pH sensitive membrane anchor for the linkage of IgG to the membrane of lipid vesicles, adherent and non-adherent cells.

Membrane Binding of HIV-1 Matrix Protein: Dependence on Bilayer Composition and Protein Lipidation

PubMed Central

Barros, Marilia; Nanda, Hirsh

2016-01-01

ABSTRACT By assembling in a protein lattice on the host's plasma membrane, the retroviral Gag polyprotein triggers formation of the viral protein/membrane shell. The MA domain of Gag employs multiple signals—electrostatic, hydrophobic, and lipid-specific—to bring the protein to the plasma membrane, thereby complementing protein-protein interactions, located in full-length Gag, in lattice formation. We report the interaction of myristoylated and unmyristoylated HIV-1 Gag MA domains with bilayers composed of purified lipid components to dissect these complex membrane signals and quantify their contributions to the overall interaction. Surface plasmon resonance on well-defined planar membrane models is used to quantify binding affinities and amounts of protein and yields free binding energy contributions, ΔG, of the various signals. Charge-charge interactions in the absence of the phosphatidylinositide PI(4,5)P2 attract the protein to acidic membrane surfaces, and myristoylation increases the affinity by a factor of 10; thus, our data do not provide evidence for a PI(4,5)P2 trigger of myristate exposure. Lipid-specific interactions with PI(4,5)P2, the major signal lipid in the inner plasma membrane, increase membrane attraction at a level similar to that of protein lipidation. While cholesterol does not directly engage in interactions, it augments protein affinity strongly by facilitating efficient myristate insertion and PI(4,5)P2 binding. We thus observe that the isolated MA protein, in the absence of protein-protein interaction conferred by the full-length Gag, binds the membrane with submicromolar affinities. IMPORTANCE Like other retroviral species, the Gag polyprotein of HIV-1 contains three major domains: the N-terminal, myristoylated MA domain that targets the protein to the plasma membrane of the host; a central capsid-forming domain; and the C-terminal, genome-binding nucleocapsid domain. These domains act in concert to condense Gag into a membrane-bounded protein lattice that recruits genomic RNA into the virus and forms the shell of a budding immature viral capsid. In binding studies of HIV-1 Gag MA to model membranes with well-controlled lipid composition, we dissect the multiple interactions of the MA domain with its target membrane. This results in a detailed understanding of the thermodynamic aspects that determine membrane association, preferential lipid recruitment to the viral shell, and those aspects of Gag assembly into the membrane-bound protein lattice that are determined by MA. PMID:26912608

Analysis of Ethylene Receptors: Ethylene-Binding Assays.

PubMed

Binder, Brad M; Schaller, G Eric

2017-01-01

Plant ethylene receptors bind ethylene with high affinity. Most of the characterization of ethylene binding to the receptors has been carried out using a radioligand-binding assay on functional receptors expressed in yeast. In this chapter, we describe methods for expressing ethylene receptors in yeast and conducting ethylene-binding assays on intact yeast and yeast membranes. The ethylene-binding assays can be modified to analyze ethylene binding to intact plants and other organisms as well as membranes isolated from any biological source.

Formation and Maturation of Phase Separated Liquid Droplets by RNA Binding Proteins

PubMed Central

Lin, Yuan; Protter, David S. W.; Rosen, Michael K.; Parker, Roy

2015-01-01

Eukaryotic cells possess numerous dynamic membrane-less organelles, RNP granules, enriched in RNA and RNA binding proteins containing disordered regions. We demonstrate that the disordered regions of key RNP granule components, and the full-length granule protein hnRNPA1, can phase separate in vitro, producing dynamic liquid droplets. Phase separation is promoted by low salt concentrations or RNA. Over time, the droplets mature to more stable states, as assessed by slowed fluorescence recovery after photobleaching and resistance to salt. Maturation often coincides with formation of fibrous structures. Different disordered domains can co-assemble into phase-separated droplets. These biophysical properties demonstrate a plausible mechanism by which interactions between disordered regions, coupled with RNA binding, could contribute to RNP granule assembly in vivo through promoting phase separation. Progression from dynamic liquids to stable fibers may be regulated to produce cellular structures with diverse physiochemical properties and functions. Misregulation could contribute to diseases involving aberrant RNA granules. PMID:26412307

Topographical analysis of the plasma membrane-associated sucrose binding protein from soybean.

PubMed

Overvoorde, P J; Grimes, H D

1994-05-27

Plasma membranes of soybean cells actively engaged in sucrose transport have a sucrose binding protein (SBP) that does not appear to be an integral membrane protein. Experiments were undertaken to analyze the topographical association of this protein with the membrane. Treatment of purified plasma membrane vesicles with either 1 M KCl or KI released less than 35% of the sucrose binding protein from the membrane whereas treatment with either 4 M urea or 0.1 M Na2CO3, pH 11.5, disassociated between 50 and 70%, respectively, of this protein from the membrane. SDS, at either 0.5x, 1x, or 10x of its critical micelle concentration, effectively solubilized the sucrose binding protein. The nonionic detergents Triton X-100 and CHAPS, at either 0.5x, 1x, or 10x of their critical micelle concentration, solubilized between 65 and 75% of this protein. When either native plasma membrane-associated or in vitro-transcribed and -translated SBP were subjected to Triton X-114 phase separation, 80% partitioned into the detergent-poor aqueous phase. These results indicate that the SBP is a peripheral membrane protein but also suggest that there is a population of this protein that is tethered to the membrane.

Binding of anti-basement membrane antibody to alveolar basement membrane after intratracheal gasoline instillation in rabbits.

PubMed Central

Yamamoto, T.; Wilson, C. B.

1987-01-01

A possible causal relationship has been suggested between hydrocarbon (gasoline, solvents, etc.) exposure and development of anti-basement membrane antibody-associated Goodpasture's syndrome in man. The authors evaluated the effect of hydrocarbons on pulmonary capillary permeability and binding of heterologous anti-basement membrane antibodies in the lungs after intratracheal instillation of minute amounts of unleaded gasoline into rabbits. The anti-glomerular basement membrane (GBM) antibodies used reacted with the alveolar basement membrane (ABM) in vitro by indirect immunofluorescence. The gasoline treatment altered pulmonary capillary permeability, judging from the increased accumulation of systemically administered radioiodinated bovine serum albumin in the alveolar and extravascular spaces of lungs; it also induced focal macroscopic and microscopic pulmonary histologic lesions. The gasoline caused focal in vivo binding of the anti-GBM antibodies to the ABM detectable by immunofluorescence microscopy. No binding was observed in lungs from control rabbits given saline instillations when assayed by immunofluorescence. The paired label radioisotope technique confirmed the increased antibody binding to lungs injured with gasoline (1.08 +/- 0.03 micrograms) versus 0.37 +/- 0.07 microgram after saline (P less than 0.001). These results indicate that gasoline exposure damages a pulmonary barrier that normally prevents binding of anti-GBM/ABM antibody to ABM and suggest that hydrocarbon exposure may be one of perhaps several pneumotoxic events that contribute to the episodic pulmonary hemorrhage in Goodpasture's syndrome by temporarily allowing ABM binding of anti-basement membrane antibodies. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:3548409

Structural basis of RND-type multidrug exporters

PubMed Central

Yamaguchi, Akihito; Nakashima, Ryosuke; Sakurai, Keisuke

2015-01-01

Bacterial multidrug exporters are intrinsic membrane transporters that act as cellular self-defense mechanism. The most notable characteristics of multidrug exporters is that they export a wide range of drugs and toxic compounds. The overexpression of these exporters causes multidrug resistance. Multidrug-resistant pathogens have become a serious problem in modern chemotherapy. Over the past decade, investigations into the structure of bacterial multidrug exporters have revealed the multidrug recognition and export mechanisms. In this review, we primarily discuss RND-type multidrug exporters particularly AcrAB-TolC, major drug exporter in Gram-negative bacteria. RND-type drug exporters are tripartite complexes comprising a cell membrane transporter, an outer membrane channel and an adaptor protein. Cell membrane transporters and outer membrane channels are homo-trimers; however, there is no consensus on the number of adaptor proteins in these tripartite complexes. The three monomers of a cell membrane transporter have varying conformations (access, binding, and extrusion) during transport. Drugs are exported following an ordered conformational change in these three monomers, through a functional rotation mechanism coupled with the proton relay cycle in ion pairs, which is driven by proton translocation. Multidrug recognition is based on a multisite drug-binding mechanism, in which two voluminous multidrug-binding pockets in cell membrane exporters recognize a wide range of substrates as a result of permutations at numerous binding sites that are specific for the partial structures of substrate molecules. The voluminous multidrug-binding pocket may have numerous binding sites even for a single substrate, suggesting that substrates may move between binding sites during transport, an idea named as multisite-drug-oscillation hypothesis. This hypothesis is consistent with the apparently broad substrate specificity of cell membrane exporters and their highly efficient ejection of drugs from the cell. Substrates are transported through dual multidrug-binding pockets via the peristaltic motion of the substrate translocation channel. Although there are no clinically available inhibitors of bacterial multidrug exporters, efforts to develop inhibitors based on structural information are underway. PMID:25941524

Structural basis of RND-type multidrug exporters.

PubMed

Yamaguchi, Akihito; Nakashima, Ryosuke; Sakurai, Keisuke

2015-01-01

Bacterial multidrug exporters are intrinsic membrane transporters that act as cellular self-defense mechanism. The most notable characteristics of multidrug exporters is that they export a wide range of drugs and toxic compounds. The overexpression of these exporters causes multidrug resistance. Multidrug-resistant pathogens have become a serious problem in modern chemotherapy. Over the past decade, investigations into the structure of bacterial multidrug exporters have revealed the multidrug recognition and export mechanisms. In this review, we primarily discuss RND-type multidrug exporters particularly AcrAB-TolC, major drug exporter in Gram-negative bacteria. RND-type drug exporters are tripartite complexes comprising a cell membrane transporter, an outer membrane channel and an adaptor protein. Cell membrane transporters and outer membrane channels are homo-trimers; however, there is no consensus on the number of adaptor proteins in these tripartite complexes. The three monomers of a cell membrane transporter have varying conformations (access, binding, and extrusion) during transport. Drugs are exported following an ordered conformational change in these three monomers, through a functional rotation mechanism coupled with the proton relay cycle in ion pairs, which is driven by proton translocation. Multidrug recognition is based on a multisite drug-binding mechanism, in which two voluminous multidrug-binding pockets in cell membrane exporters recognize a wide range of substrates as a result of permutations at numerous binding sites that are specific for the partial structures of substrate molecules. The voluminous multidrug-binding pocket may have numerous binding sites even for a single substrate, suggesting that substrates may move between binding sites during transport, an idea named as multisite-drug-oscillation hypothesis. This hypothesis is consistent with the apparently broad substrate specificity of cell membrane exporters and their highly efficient ejection of drugs from the cell. Substrates are transported through dual multidrug-binding pockets via the peristaltic motion of the substrate translocation channel. Although there are no clinically available inhibitors of bacterial multidrug exporters, efforts to develop inhibitors based on structural information are underway.

Plasma membrane order and fluidity are diversely triggered by elicitors of plant defence

PubMed Central

Sandor, Roman; Der, Christophe; Grosjean, Kevin; Anca, Iulia; Noirot, Elodie; Leborgne-Castel, Nathalie; Lochman, Jan; Simon-Plas, Françoise; Gerbeau-Pissot, Patricia

2016-01-01

Although plants are exposed to a great number of pathogens, they usually defend themselves by triggering mechanisms able to limit disease development. Alongside signalling events common to most such incompatible interactions, modifications of plasma membrane (PM) physical properties could be new players in the cell transduction cascade. Different pairs of elicitors (cryptogein, oligogalacturonides, and flagellin) and plant cells (tobacco and Arabidopsis) were used to address the issue of possible modifications of plant PM biophysical properties induced by elicitors and their links to other events of the defence signalling cascade. We observed an increase of PM order whatever the elicitor/plant cell pair used, provided that a signalling cascade was induced. Such membrane modification is dependent on the NADPH oxidase-mediated reactive oxygen species production. Moreover, cryptogein, which is the sole elicitor able to trap sterols, is also the only one able to trigger an increase in PM fluidity. The use of cryptogein variants with altered sterol-binding properties confirms the strong correlation between sterol removal from the PM and PM fluidity enhancement. These results propose PM dynamics as a player in early signalling processes triggered by elicitors of plant defence. PMID:27604805

Effect of myristoylated N-terminus of Arf1 on the bending rigidity of phospholipid membranes

NASA Astrophysics Data System (ADS)

Burrola Gabilondo, Beatriz; Zhou, Hernan; Randazzo, Paul A.; Losert, Wolfgang

2010-03-01

The protein Arf1 is part of the COPI vesicle transport process from the Golgi to the ER. It binds to membranes via a myristoylated N-terminus and it has been shown to tubulate Large Unilamellar Vesicles. The effect of the N-terminus of Arf1 on physical properties of membranes has not been studied, with the exception of curvature. We previously found that the myristoylated N-terminus increases the packing of the lipid molecules, but has no effect on the lateral mobility. We tested the hypothesis that myristoylated peptides affect the bending rigidity of phospholipid Giant Unilamellar Vesicles (GUV). We use optical tweezers to pull tethers from GUV and measure the force of pulling the tether, as well as the retraction speed of the tether once it is released. We also used flicker spectroscopy to estimate the values of the mechanical properties of GUV. We will present results of the force and tether retraction measurements, as well as mechanical properties estimates from flicker, for GUV in the presence of varying concentrations of myristoylated and non-myristoylated N-terminus of Arf1, and compare these with measurements for GUV in the absence of peptide.

Membrane proteins bind lipids selectively to modulate their structure and function.

PubMed

Laganowsky, Arthur; Reading, Eamonn; Allison, Timothy M; Ulmschneider, Martin B; Degiacomi, Matteo T; Baldwin, Andrew J; Robinson, Carol V

2014-06-05

Previous studies have established that the folding, structure and function of membrane proteins are influenced by their lipid environments and that lipids can bind to specific sites, for example, in potassium channels. Fundamental questions remain however regarding the extent of membrane protein selectivity towards lipids. Here we report a mass spectrometry approach designed to determine the selectivity of lipid binding to membrane protein complexes. We investigate the mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis and aquaporin Z (AqpZ) and the ammonia channel (AmtB) from Escherichia coli, using ion mobility mass spectrometry (IM-MS), which reports gas-phase collision cross-sections. We demonstrate that folded conformations of membrane protein complexes can exist in the gas phase. By resolving lipid-bound states, we then rank bound lipids on the basis of their ability to resist gas phase unfolding and thereby stabilize membrane protein structure. Lipids bind non-selectively and with high avidity to MscL, all imparting comparable stability; however, the highest-ranking lipid is phosphatidylinositol phosphate, in line with its proposed functional role in mechanosensation. AqpZ is also stabilized by many lipids, with cardiolipin imparting the most significant resistance to unfolding. Subsequently, through functional assays we show that cardiolipin modulates AqpZ function. Similar experiments identify AmtB as being highly selective for phosphatidylglycerol, prompting us to obtain an X-ray structure in this lipid membrane-like environment. The 2.3 Å resolution structure, when compared with others obtained without lipid bound, reveals distinct conformational changes that re-position AmtB residues to interact with the lipid bilayer. Our results demonstrate that resistance to unfolding correlates with specific lipid-binding events, enabling a distinction to be made between lipids that merely bind from those that modulate membrane protein structure and/or function. We anticipate that these findings will be important not only for defining the selectivity of membrane proteins towards lipids, but also for understanding the role of lipids in modulating protein function or drug binding.

STARD4 Membrane Interactions and Sterol Binding

PubMed Central

2016-01-01

The steroidogenic acute regulatory protein-related lipid transfer (START) domain family is defined by a conserved 210-amino acid sequence that folds into an α/β helix-grip structure. Members of this protein family bind a variety of ligands, including cholesterol, phospholipids, sphingolipids, and bile acids, with putative roles in nonvesicular lipid transport, metabolism, and cell signaling. Among the soluble START proteins, STARD4 is expressed in most tissues and has previously been shown to transfer sterol, but the molecular mechanisms of membrane interaction and sterol binding remain unclear. In this work, we use biochemical techniques to characterize regions of STARD4 and determine their role in membrane interaction and sterol binding. Our results show that STARD4 interacts with anionic membranes through a surface-exposed basic patch and that introducing a mutation (L124D) into the Omega-1 (Ω1) loop, which covers the sterol binding pocket, attenuates sterol transfer activity. To gain insight into the attenuating mechanism of the L124D mutation, we conducted structural and biophysical studies of wild-type and L124D STARD4. These studies show that the L124D mutation reduces the conformational flexibility of the protein, resulting in a diminished level of membrane interaction and sterol transfer. These studies also reveal that the C-terminal α-helix, and not the Ω1 loop, partitions into the membrane bilayer. On the basis of these observations, we propose a model of STARD4 membrane interaction and sterol binding and release that requires dynamic movement of both the Ω1 loop and membrane insertion of the C-terminal α-helix. PMID:26168008

Structural Sensitivity of a Prokaryotic Pentameric Ligand-gated Ion Channel to Its Membrane Environment*

PubMed Central

Labriola, Jonathan M.; Pandhare, Akash; Jansen, Michaela; Blanton, Michael P.; Corringer, Pierre-Jean; Baenziger, John E.

2013-01-01

Although the activity of the nicotinic acetylcholine receptor (nAChR) is exquisitely sensitive to its membrane environment, the underlying mechanisms remain poorly defined. The homologous prokaryotic pentameric ligand-gated ion channel, Gloebacter ligand-gated ion channel (GLIC), represents an excellent model for probing the molecular basis of nAChR sensitivity because of its high structural homology, relative ease of expression, and amenability to crystallographic analysis. We show here that membrane-reconstituted GLIC exhibits structural and biophysical properties similar to those of the membrane-reconstituted nAChR, although GLIC is substantially more thermally stable. GLIC, however, does not possess the same exquisite lipid sensitivity. In particular, GLIC does not exhibit the same propensity to adopt an uncoupled conformation where agonist binding is uncoupled from channel gating. Structural comparisons provide insight into the chemical features that may predispose the nAChR to the formation of an uncoupled state. PMID:23463505

Cooperative binding of Annexin A5 to phosphatidylserine on apoptotic cell membranes

NASA Astrophysics Data System (ADS)

Janko, Christina; Jeremic, Ivica; Biermann, Mona; Chaurio, Ricardo; Schorn, Christine; Muñoz, Luis E.; Herrmann, Martin

2013-12-01

Healthy cells exhibit an asymmetric plasma membrane with phosphatidylserine (PS) located on the cytoplasmic leaflet of the plasma membrane bilayer. Annexin A5-FITC, a PS binding protein, is commonly used to evaluate apoptosis in flow cytometry. PS exposed by apoptotic cells serves as a major ‘eat-me’ signal for phagocytes. Although exposition of PS has been observed after alternative stimuli, no clearance of viable, PS exposing cells has been detected. Thus, besides PS exposure, membranes of viable and apoptotic cells might exhibit specific characteristics. Here, we show that Annexin A5 binds in a cooperative manner to different types of dead cells. Shrunken apoptotic cells thereby showed the highest Hill coefficient values. Contrarily, parafomaldehyde fixation of apoptotic cells completely abrogates the cooperativity effect seen with dead and dying cells. We tend to speculate that the cooperative binding of Annexin A5 to the membranes of apoptotic cells reflects higher fluidity of the exposed membranes facilitating PS clustering.

Membrane Stored Curvature Elastic Stress Modulates Recruitment of Maintenance Proteins PspA and Vipp1

PubMed Central

McDonald, Christopher; Jovanovic, Goran; Ces, Oscar

2015-01-01

ABSTRACT Phage shock protein A (PspA), which is responsible for maintaining inner membrane integrity under stress in enterobacteria, and vesicle-inducting protein in plastids 1 (Vipp1), which functions for membrane maintenance and thylakoid biogenesis in cyanobacteria and plants, are similar peripheral membrane-binding proteins. Their homologous N-terminal amphipathic helices are required for membrane binding; however, the membrane features recognized and required for expressing their functionalities have remained largely uncharacterized. Rigorously controlled, in vitro methodologies with lipid vesicles and purified proteins were used in this study and provided the first biochemical and biophysical characterizations of membrane binding by PspA and Vipp1. Both proteins are found to sense stored curvature elastic (SCE) stress and anionic lipids within the membrane. PspA has an enhanced sensitivity for SCE stress and a higher affinity for the membrane than Vipp1. These variations in binding may be crucial for some of the proteins’ differing roles in vivo. Assays probing the transcriptional regulatory function of PspA in the presence of vesicles showed that a relief of transcription inhibition occurs in an SCE stress-specific manner. This in vitro recapitulation of membrane stress-dependent transcription control suggests that the Psp response may be mounted in vivo when a cell’s inner membrane experiences increased SCE stress. PMID:26330516

Channel formation by the binding component of Clostridium botulinum C2 toxin: glutamate 307 of C2II affects channel properties in vitro and pH-dependent C2I translocation in vivo.

PubMed

Blöcker, Dagmar; Bachmeyer, Christoph; Benz, Roland; Aktories, Klaus; Barth, Holger

2003-05-13

The binding component (C2II) of the binary Clostridium botulinum C2 toxin mediates transport of the actin ADP-ribosylating enzyme component (C2I) into the cytosol of target cells. C2II (80 kDa) is activated by trypsin cleavage, and proteolytically activated C2II (60 kDa) oligomerizes to heptamers in solution. Activated C2II forms channels in lipid bilayer membranes which are highly cation selective and voltage-gated. A role for this channel in C2I translocation across the cell membrane into the cytosol is discussed. Amino acid residues 303-331 of C2II contain a conserved pattern of alternating hydrophobic and hydrophilic residues, which likely facilitates membrane insertion and channel formation by creating two antiparallel beta-strands. Some of the residues are in strategic positions within the putative C2II channel, in particular, glutamate 307 (E307) localized in its center and glycine 316 (G316) localized on the trans side of the membrane. Here, single-lysine substitutions of these amino acids and the double mutant E307K/G316K of C2II were analyzed in vivo and in artificial lipid bilayer experiments. The pH dependence of C2I transport across cellular membranes was altered, and a pH of

Binding of canonical Wnt ligands to their receptor complexes occurs in ordered plasma membrane environments.

PubMed

Sezgin, Erdinc; Azbazdar, Yagmur; Ng, Xue W; Teh, Cathleen; Simons, Kai; Weidinger, Gilbert; Wohland, Thorsten; Eggeling, Christian; Ozhan, Gunes

2017-08-01

While the cytosolic events of Wnt/β-catenin signaling (canonical Wnt signaling) pathway have been widely studied, only little is known about the molecular mechanisms involved in Wnt binding to its receptors at the plasma membrane. Here, we reveal the influence of the immediate plasma membrane environment on the canonical Wnt-receptor interaction. While the receptors are distributed both in ordered and disordered environments, Wnt binding to its receptors selectively occurs in more ordered membrane environments which appear to cointernalize with the Wnt-receptor complex. Moreover, Wnt/β-catenin signaling is significantly reduced when the membrane order is disturbed by specific inhibitors of certain lipids that prefer to localize at the ordered environments. Similarly, a reduction in Wnt signaling activity is observed in Niemann-Pick Type C disease cells where trafficking of ordered membrane lipid components to the plasma membrane is genetically impaired. We thus conclude that ordered plasma membrane environments are essential for binding of canonical Wnts to their receptor complexes and downstream signaling activity. © 2017 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

Salt Bridge Formation between the I-BAR Domain and Lipids Increases Lipid Density and Membrane Curvature.

PubMed

Takemura, Kazuhiro; Hanawa-Suetsugu, Kyoko; Suetsugu, Shiro; Kitao, Akio

2017-07-28

The BAR domain superfamily proteins sense or induce curvature in membranes. The inverse-BAR domain (I-BAR) is a BAR domain that forms a straight "zeppelin-shaped" dimer. The mechanisms by which IRSp53 I-BAR binds to and deforms a lipid membrane are investigated here by all-atom molecular dynamics simulation (MD), binding energy analysis, and the effects of mutation experiments on filopodia on HeLa cells. I-BAR adopts a curved structure when crystallized, but adopts a flatter shape in MD. The binding of I-BAR to membrane was stabilized by ~30 salt bridges, consistent with experiments showing that point mutations of the interface residues have little effect on the binding affinity whereas multiple mutations have considerable effect. Salt bridge formation increases the local density of lipids and deforms the membrane into a concave shape. In addition, the point mutations that break key intra-molecular salt bridges within I-BAR reduce the binding affinity; this was confirmed by expressing these mutants in HeLa cells and observing their effects. The results indicate that the stiffness of I-BAR is important for membrane deformation, although I-BAR does not act as a completely rigid template.

Interaction between two adapter proteins, PAG and EBP50: a possible link between membrane rafts and actin cytoskeleton.

PubMed

Brdicková, N; Brdicka, T; Andera, L; Spicka, J; Angelisová, P; Milgram, S L; Horejsí, V

2001-10-26

Phosphoprotein associated with GEMs (PAG), also known as Csk-binding protein (Cbp), is a broadly expressed palmitoylated transmembrane adapter protein found in membrane rafts, also called GEMs (glycosphingolipid-enriched membrane microdomains). PAG is known to bind and activate the essential regulator of Src-family kinases, cytoplasmic protein tyrosine kinase Csk. In the present study we used the yeast 2-hybrid system to search for additional proteins which might bind to PAG. We have identified the abundant cytoplasmic adapter protein EBP50 (ezrin/radixin/moesin (ERM)-binding phosphoprotein of 50 kDa), also known as NHERF (Na(+)/H(+) exchanger regulatory factor), as a specific PAG-binding partner. The interaction involves the C-terminal sequence (TRL) of PAG and N-terminal PDZ domain(s) of EBP50. As EBP50 is known to interact via its C-terminal domain with the ERM-family proteins, which in turn bind to actin cytoskeleton, the PAG-EBP50 interaction may be important for connecting membrane rafts to the actin cytoskeleton.

Functional properties of an isolated. cap alpha beta. heterodimeric human placenta insulin-like growth factor 1 receptor complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Feltz, S.M.; Swanson, M.L.; Wemmie, J.A.

1988-05-03

Treatment of human placenta membranes at pH 8.5 in the presence of 2.0 mM dithiothreitol (DTT) for 5 min, followed by the simultaneous removal of the DTT and pH adjustment of pH 7.6, resulted in the formation of a functional ..cap alpha beta.. heterodimeric insulin-like growth factor 1 (IGF-1) receptor complex from the native ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric disulfide-linked state. The membrane-bound ..cap alpha beta.. heterodimeric complex displayed similar curvilinear /sup 125/I-IGF-1 equilibrium binding compared to the ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric complex. /sup 125/I-IGF-1 binding to both the isolated ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric and ..cap alpha beta..more » heterodimeric complexes demonstrated a marked straightening of the Scatchard plots, compared to the placenta membrane-bound IGF-1 receptors, with a 2-fold increase in the high-affinity binding component. IGF-1 stimulation of IGF-1 receptor autophosphorylation indicated that the ligand-dependent activation of ..cap alpha beta.. heterodimeric protein kinase activity occurred concomitant with the reassociation into a covalent ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric state. These data demonstrate that (i) a combination of alkaline pH and DTT treatment of human placenta membranes results in the formation of an ..cap alpha beta.. heterodimeric IGF-1 receptor complex, (ii) unlike the insulin receptor, high-affinity homogeneous IGF-1 binding occurs in both the ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric and ..cap alpha beta.. heterodimeric complexes, and (iii) IGF-1-dependent autophosphorylation of the ..cap alpha beta.. heterodimeric IGF-1 receptor complex correlates wit an IGF-1 dependent covalent reassociation into an ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric disulfide-linked state.« less

cAMP regulates DEP domain-mediated binding of the guanine nucleotide exchange factor Epac1 to phosphatidic acid at the plasma membrane.

PubMed

Consonni, Sarah V; Gloerich, Martijn; Spanjaard, Emma; Bos, Johannes L

2012-03-06

Epac1 is a cAMP-regulated guanine nucleotide exchange factor for the small G protein Rap. Upon cAMP binding, Epac1 undergoes a conformational change that results in its release from autoinhibition. In addition, cAMP induces the translocation of Epac1 from the cytosol to the plasma membrane. This relocalization of Epac1 is required for efficient activation of plasma membrane-located Rap and for cAMP-induced cell adhesion. This translocation requires the Dishevelled, Egl-10, Pleckstrin (DEP) domain, but the molecular entity that serves as the plasma membrane anchor and the possible mechanism of regulated binding remains elusive. Here we show that Epac1 binds directly to phosphatidic acid. Similar to the cAMP-induced Epac1 translocation, this binding is regulated by cAMP and requires the DEP domain. Furthermore, depletion of phosphatidic acid by inhibition of phospholipase D1 prevents cAMP-induced translocation of Epac1 as well as the subsequent activation of Rap at the plasma membrane. Finally, mutation of a single basic residue within a polybasic stretch of the DEP domain, which abolishes translocation, also prevents binding to phosphatidic acid. From these results we conclude that cAMP induces a conformational change in Epac1 that enables DEP domain-mediated binding to phosphatidic acid, resulting in the tethering of Epac1 at the plasma membrane and subsequent activation of Rap.

Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases.

PubMed

Jadwin, Joshua A; Oh, Dongmyung; Curran, Timothy G; Ogiue-Ikeda, Mari; Jia, Lin; White, Forest M; Machida, Kazuya; Yu, Ji; Mayer, Bruce J

2016-04-12

While the affinities and specificities of SH2 domain-phosphotyrosine interactions have been well characterized, spatio-temporal changes in phosphosite availability in response to signals, and their impact on recruitment of SH2-containing proteins in vivo, are not well understood. To address this issue, we used three complementary experimental approaches to monitor phosphorylation and SH2 binding in human A431 cells stimulated with epidermal growth factor (EGF): 1) phospho-specific mass spectrometry; 2) far-Western blotting; and 3) live cell single-molecule imaging of SH2 membrane recruitment. Far-Western and MS analyses identified both well-established and previously undocumented EGF-dependent tyrosine phosphorylation and binding events, as well as dynamic changes in binding patterns over time. In comparing SH2 binding site phosphorylation with SH2 domain membrane recruitment in living cells, we found in vivo binding to be much slower. Delayed SH2 domain recruitment correlated with clustering of SH2 domain binding sites on the membrane, consistent with membrane retention via SH2 rebinding.

Coarse-Grained Molecular Dynamics Simulations of Membrane-Trehalose Interactions.

PubMed

Kapla, Jon; Stevensson, Baltzar; Maliniak, Arnold

2016-09-15

It is well established that trehalose (TRH) affects the physical properties of lipid bilayers and stabilizes biological membranes. We present molecular dynamics (MD) computer simulations to investigate the interactions between lipid membranes formed by 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and TRH. Both atomistic and coarse-grained (CG) interaction models were employed, and the coarse graining of DMPC leads to a reduction in the acyl chain length corresponding to a 1,2-dilauroyl-sn-glycero-3-phosphocholine lipid (DLPC). Several modifications of the Martini interaction model, used for CG simulations, were implemented, resulting in different potentials of mean force (PMFs) for DMPC bilayer-TRH interactions. These PMFs were subsequently used in a simple two-site analytical model for the description of sugar binding at the membrane interface. In contrast to that in atomistic MD simulations, the binding in the CG model was not in agreement with the two-site model. Our interpretation is that the interaction balance, involving water, TRH, and lipids, in the CG systems needs further tuning of the force-field parameters. The area per lipid is only weakly affected by TRH concentration, whereas the compressibility modulus related to the fluctuations of the membrane increases with an increase in TRH content. In agreement with experimental findings, the bending modulus is not affected by the inclusion of TRH. The important aspects of lipid bilayer interactions with biomolecules are membrane curvature generation and sensing. In the present investigation, membrane curvature is generated by artificial buckling of the bilayer in one dimension. It turns out that TRH prefers the regions with the highest curvature, which enables the most favorable situation for lipid-sugar interactions.

A BAR domain in the N terminus of the Arf GAP ASAP1 affects membrane structure and trafficking of epidermal growth factor receptor.

PubMed

Nie, Zhongzhen; Hirsch, Dianne S; Luo, Ruibai; Jian, Xiaoying; Stauffer, Stacey; Cremesti, Aida; Andrade, Josefa; Lebowitz, Jacob; Marino, Michael; Ahvazi, Bijan; Hinshaw, Jenny E; Randazzo, Paul A

2006-01-24

Arf GAPs are multidomain proteins that function in membrane traffic by inactivating the GTP binding protein Arf1. Numerous Arf GAPs contain a BAR domain, a protein structural element that contributes to membrane traffic by either inducing or sensing membrane curvature. We have examined the role of a putative BAR domain in the function of the Arf GAP ASAP1. ASAP1's N terminus, containing the putative BAR domain together with a PH domain, dimerized to form an extended structure that bound to large unilamellar vesicles containing acidic phospholipids, properties that define a BAR domain. A recombinant protein containing the BAR domain of ASAP1, together with the PH and Arf GAP domains, efficiently bent the surface of large unilamellar vesicles, resulting in the formation of tubular structures. This activity was regulated by Arf1*GTP binding to the Arf GAP domain. In vivo, the tubular structures induced by ASAP1 mutants contained epidermal growth factor receptor (EGFR) and Rab11, and ASAP1 colocalized in tubular structures with EGFR during recycling of receptor. Expression of ASAP1 accelerated EGFR trafficking and slowed cell spreading. An ASAP1 mutant lacking the BAR domain had no effect. The N-terminal BAR domain of ASAP1 mediates membrane bending and is necessary for ASAP1 function. The Arf dependence of the bending activity is consistent with ASAP1 functioning as an Arf effector.

The Arabidopsis COBRA Protein Facilitates Cellulose Crystallization at the Plasma Membrane*

PubMed Central

Sorek, Nadav; Sorek, Hagit; Kijac, Aleksandra; Szemenyei, Heidi J.; Bauer, Stefan; Hématy, Kian; Wemmer, David E.; Somerville, Chris R.

2014-01-01

Mutations in the Arabidopsis COBRA gene lead to defects in cellulose synthesis but the function of COBRA is unknown. Here we present evidence that COBRA localizes to discrete particles in the plasma membrane and is sensitive to inhibitors of cellulose synthesis, suggesting that COBRA and the cellulose synthase complex reside in close proximity on the plasma membrane. Live-cell imaging of cellulose synthesis indicated that, once initiated, cellulose synthesis appeared to proceed normally in the cobra mutant. Using isothermal calorimetry, COBRA was found to bind individual β1–4-linked glucan chains with a KD of 3.2 μm. Competition assays suggests that COBRA binds individual β1–4-linked glucan chains with higher affinity than crystalline cellulose. Solid-state nuclear magnetic resonance studies of the cell wall of the cobra mutant also indicated that, in addition to decreases in cellulose amount, the properties of the cellulose fibrils and other cell wall polymers differed from wild type by being less crystalline and having an increased number of reducing ends. We interpret the available evidence as suggesting that COBRA facilitates cellulose crystallization from the emerging β1–4-glucan chains by acting as a “polysaccharide chaperone.” PMID:25331944

Analysis of the synaptotagmin family during reconstituted membrane fusion. Uncovering a class of inhibitory isoforms.

PubMed

Bhalla, Akhil; Chicka, Michael C; Chapman, Edwin R

2008-08-01

Ca(2+)-triggered exocytosis in neurons and neuroendocrine cells is regulated by the Ca(2+)-binding protein synaptotagmin (syt) I. Sixteen additional isoforms of syt have been identified, but little is known concerning their biochemical or functional properties. Here, we assessed the abilities of fourteen syt isoforms to directly regulate SNARE (soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor)-catalyzed membrane fusion. One group of isoforms stimulated neuronal SNARE-mediated fusion in response to Ca(2+), while another set inhibited SNARE catalyzed fusion in both the absence and presence of Ca(2+). Biochemical analysis revealed a strong correlation between the ability of syt isoforms to bind 1,2-dioleoyl phosphatidylserine (PS) and t-SNAREs in a Ca(2+)-promoted manner with their abilities to enhance fusion, further establishing PS and SNAREs as critical effectors for syt action. The ability of syt I to efficiently stimulate fusion was specific for certain SNARE pairs, suggesting that syts might contribute to the specificity of intracellular membrane fusion reactions. Finally, a subset of inhibitory syts down-regulated the ability of syt I to activate fusion, demonstrating that syt isoforms can modulate the function of each other.

Direct labelling of the human P2X7 receptor and identification of positive and negative cooperativity of binding.

PubMed

Michel, A D; Chambers, L J; Clay, W C; Condreay, J P; Walter, D S; Chessell, I P

2007-05-01

The P2X(7) receptor exhibits complex pharmacological properties. In this study, binding of a [(3)H]-labelled P2X(7) receptor antagonist to human P2X(7) receptors has been examined to further understand ligand interactions with this receptor. The P2X(7) receptor antagonist, N-[2-({2-[(2-hydroxyethyl)amino]ethyl}amino)-5-quinolinyl]-2-tricyclo[3.3.1.1(3,7)]dec-1-ylacetamide (compound-17), was radiolabelled with tritium and binding studies were performed using membranes prepared from U-2 OS or HEK293 cells expressing human recombinant P2X(7) receptors. Binding of [(3)H]-compound-17 was higher in membranes prepared from cells expressing P2X(7) receptors than from control cells and was inhibited by ATP suggesting labelled sites represented human P2X(7) receptors. Binding was reversible, saturable and modulated by P2X(7) receptor ligands (Brilliant Blue G, KN62, ATP, decavanadate). Furthermore, ATP potency was reduced in the presence of divalent cations or NaCl. Radioligand binding exhibited both positive and negative cooperativity. Positive cooperativity was evident from bell shaped Scatchard plots, reduction in radioligand dissociation rate by unlabelled compound-17 and enhancement of radioligand binding by KN62 and unlabelled compound-17. ATP and decavanadate inhibited binding in a negative cooperative manner as they enhanced radioligand dissociation. These data demonstrate that human P2X(7) receptors can be directly labelled and provide novel insights into receptor function. The positive cooperativity observed suggests that binding of compound-17 to one subunit in the P2X(7) receptor complex enhances subsequent binding to other P2X(7) subunits in the same complex. The negative cooperative effects of ATP suggest that ATP and compound-17 bind at separate, interacting, sites on the P2X(7) receptor.

Direct labelling of the human P2X7 receptor and identification of positive and negative cooperativity of binding

PubMed Central

Michel, A D; Chambers, L J; Clay, W C; Condreay, J P; Walter, D S; Chessell, I P

2007-01-01

Background and Purpose: The P2X7 receptor exhibits complex pharmacological properties. In this study, binding of a [3H]-labelled P2X7 receptor antagonist to human P2X7 receptors has been examined to further understand ligand interactions with this receptor. Experimental Approach: The P2X7 receptor antagonist, N-[2-({2-[(2-hydroxyethyl)amino]ethyl}amino)-5-quinolinyl]-2-tricyclo[3.3.1.13,7]dec-1-ylacetamide (compound-17), was radiolabelled with tritium and binding studies were performed using membranes prepared from U-2 OS or HEK293 cells expressing human recombinant P2X7 receptors. Key Results: Binding of [3H]-compound-17 was higher in membranes prepared from cells expressing P2X7 receptors than from control cells and was inhibited by ATP suggesting labelled sites represented human P2X7 receptors. Binding was reversible, saturable and modulated by P2X7 receptor ligands (Brilliant Blue G, KN62, ATP, decavanadate). Furthermore, ATP potency was reduced in the presence of divalent cations or NaCl. Radioligand binding exhibited both positive and negative cooperativity. Positive cooperativity was evident from bell shaped Scatchard plots, reduction in radioligand dissociation rate by unlabelled compound-17 and enhancement of radioligand binding by KN62 and unlabelled compound-17. ATP and decavanadate inhibited binding in a negative cooperative manner as they enhanced radioligand dissociation. Conclusions: These data demonstrate that human P2X7 receptors can be directly labelled and provide novel insights into receptor function. The positive cooperativity observed suggests that binding of compound-17 to one subunit in the P2X7 receptor complex enhances subsequent binding to other P2X7 subunits in the same complex. The negative cooperative effects of ATP suggest that ATP and compound-17 bind at separate, interacting, sites on the P2X7 receptor. PMID:17339830

Cd-binding to model membranes

NASA Astrophysics Data System (ADS)

Geszner, R.; Saibene, S.; Butz, T.; Lerf, A.

1990-08-01

The binding of Cd2+ to the model membranes Di-myristoyl L-α-phosphatidic acid (DMPA) and Di-myristoyl L-α-phosphatidylcholine (DMPC) was studied by time differential perturbed angular correlation (TDPAC) on111mCd, via its nuclear quadrupole interaction. Whereas Cd2+ does not bind to the neutral DMPC, it binds to charged DMPA up to a 0.8∶1 Cd/lipid ratio.

Fungal toxins bind to the URF13 protein in maize mitochondria and Escherichia coli.

PubMed Central

Braun, C J; Siedow, J N; Levings, C S

1990-01-01

Expression of the maize mitochondrial T-urf13 gene results in a sensitivity to a family of fungal pathotoxins and to methomyl, a structurally unrelated systemic insecticide. Similar effects of pathotoxins and methomyl are observed when T-urf13 is cloned and expressed in Escherichia coli. An interaction between these compounds and the membrane-bound URF13 protein permeabilizes the inner mitochondrial and bacterial plasma membranes. To understand the toxin-URF13 effects, we have investigated whether toxin specifically binds to the URF13 protein. Our studies indicate that toxin binds to the URF13 protein in maize mitochondria and in E. coli expressing URF13. Binding analysis in E. coli reveals cooperative toxin binding. A low level of specific toxin binding is also demonstrated in cms-T and cms-T-restored mitochondria; however, binding does not appear to be cooperative in maize mitochondria. Competition and displacement studies in E. coli demonstrate that toxin binding is reversible and that the toxins and methomyl compete for the same, or for overlapping, binding sites. Two toxin-insensitive URF13 mutants display a diminished capability to bind toxin in E. coli, which identifies residues of URF13 important in toxin binding. A third toxin-insensitive URF13 mutant shows considerable toxin binding in E. coli, demonstrating that toxin binding can occur without causing membrane permeabilization. Our results indicate that toxin-mediated membrane permeabilization only occurs when toxin or methomyl is bound to URF13. PMID:2136632

Amyotrophic lateral sclerosis-linked mutations increase the viscosity of liquid-like TDP-43 RNP granules in neurons.

PubMed

Gopal, Pallavi P; Nirschl, Jeffrey J; Klinman, Eva; Holzbaur, Erika L F

2017-03-21

Ribonucleoprotein (RNP) granules are enriched in specific RNAs and RNA-binding proteins (RBPs) and mediate critical cellular processes. Purified RBPs form liquid droplets in vitro through liquid-liquid phase separation and liquid-like non-membrane-bound structures in cells. Mutations in the human RBPs TAR-DNA binding protein 43 (TDP-43) and RNA-binding protein FUS cause amyotrophic lateral sclerosis (ALS), but the biophysical properties of these proteins have not yet been studied in neurons. Here, we show that TDP-43 RNP granules in axons of rodent primary cortical neurons display liquid-like properties, including fusion with rapid relaxation to circular shape, shear stress-induced deformation, and rapid fluorescence recovery after photobleaching. RNP granules formed from wild-type TDP-43 show distinct biophysical properties depending on axonal location, suggesting maturation to a more stabilized structure is dependent on subcellular context, including local density and aging. Superresolution microscopy demonstrates that the stabilized population of TDP-43 RNP granules in the proximal axon is less circular and shows spiculated edges, whereas more distal granules are both more spherical and more dynamic. RNP granules formed by ALS-linked mutant TDP-43 are more viscous and exhibit disrupted transport dynamics. We propose these altered properties may confer toxic gain of function and reflect differential propensity for pathological transformation.

Freeze-fracture studies of photoreceptor membranes: new observations bearing upon the distribution of cholesterol

PubMed Central

1983-01-01

We performed electron microscopy of replicas from freeze-fractured retinas exposed during or after fixation to the cholesterol-binding antibiotic, filipin. We observed characteristic filipin-induced perturbations throughout the disk and plasma membranes of retinal rod outer segments of various species. It is evident that a prolonged exposure to filipin in fixative enhances rather than reduces presumptive cholesterol detection in the vertebrate photoreceptor cell. In agreement with the pattern seen in our previous study (Andrews, L.D., and A. I. Cohen, 1979, J. Cell Biol., 81:215-228), filipin- binding in membranes exhibiting particle-free patches seemed largely confined to these patches. Favorably fractured photoreceptors exhibited marked filipin-binding in apical inner segment plasma membrane topologically confluent with and proximate to the outer segment plasma membrane, which was comparatively free of filipin binding. A possible boundary between these differing membrane domains was suggested in a number of replicas exhibiting lower filipin binding to the apical plasma membrane of the inner segment in the area surrounding the cilium. This area contains a structure (Andrews, L. D., 1982, Freeze- fracture studies of vertebrate photoreceptors, In Structure of the Eye, J. G. Hollyfield and E. Acosta Vidrio, editors, Elsevier/North-Holland, New York, 11-23) that resembles the active zones of the nerve terminals for the frog neuromuscular junction. These observations lead us to hypothesize that these structures may function to direct vesicle fusion to occur near them, in a domain of membrane more closely resembling outer than inner segment plasma membrane. The above evidence supports the views that (a) all disk membranes contain cholesterol, but the particle-free patches present in some disks trap cholesterol from contiguous particulate membrane regions; (b) contiguous inner and outer segment membranes may greatly differ in cholesterol content; and (c) the suggested higher cholesterol in the inner segment than in the outer segment plasma membrane may help direct newly inserted photopigment molecules to the outer segment. PMID:6411740

Kinase Associated-1 Domains Drive MARK/PAR1 Kinases to Membrane Targets by Binding Acidic Phospholipids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moravcevic, Katarina; Mendrola, Jeannine M.; Schmitz, Karl R.

Phospholipid-binding modules such as PH, C1, and C2 domains play crucial roles in location-dependent regulation of many protein kinases. Here, we identify the KA1 domain (kinase associated-1 domain), found at the C terminus of yeast septin-associated kinases (Kcc4p, Gin4p, and Hsl1p) and human MARK/PAR1 kinases, as a membrane association domain that binds acidic phospholipids. Membrane localization of isolated KA1 domains depends on phosphatidylserine. Using X-ray crystallography, we identified a structurally conserved binding site for anionic phospholipids in KA1 domains from Kcc4p and MARK1. Mutating this site impairs membrane association of both KA1 domains and intact proteins and reveals the importancemore » of phosphatidylserine for bud neck localization of yeast Kcc4p. Our data suggest that KA1 domains contribute to coincidence detection, allowing kinases to bind other regulators (such as septins) only at the membrane surface. These findings have important implications for understanding MARK/PAR1 kinases, which are implicated in Alzheimer's disease, cancer, and autism.« less

Solubilization of phencyclidine receptors from rat cerebral cortex in an active ligand binding site

DOE Office of Scientific and Technical Information (OSTI.GOV)

McVittie, L.D.; Sibley, D.R.

1989-01-01

A phencyclidine (PCP) receptor binding site has been solubilized in an active ligand-binding state from rat cerebral cortical membranes with sodium deoxycholate. Optimal receptor solubilization occurs at a detergent/protein ratio of 0.5 (w/w); for 5 mg protein/ml solubilized with 0.25% sodium deoxycholate, about 60% of the protein and 25% of the receptor is solubilized. Specific binding of either (/sup 3/H)-N-(1-(2-thienyl)cyclohexyl)piperidine ((/sup 3/H)TCP) or (/sup 3/H)MK-801 is measurable by filtration through Sephadex G-50 columns or glass fiber filters; more than 60% of the binding activity is stable after 48 h at 4/degrees/C. In the presence of detergent, (/sup 3/H)TCP binding exhibitsmore » a K/sub d/ of 250 nM, a B/sub max/ of 0.56 pmol/mg protein, and a pharmacological profile consistent with that of the membrane-bound PCP receptor, although most drugs bind with affinities 2 to 8 fold lower than in membranes. Upon reduction of detergent concentration, binding parameters approximate those for the membrane-bound receptor (/sup 3/H)TCP binding: K/sub d/ = 48 nM, M/sub max/ = 1.13 pmol/mg protein.« less

Determination of structure of the MinD-ATP complex reveals the orientation of MinD on the membrane and the relative location of the binding sites for MinE and MinC

PubMed Central

Wu, Wei; Park, Kyung-Tae; Holyoak, Todd; Lutkenhaus, Joe

2011-01-01

Summary The three Min proteins spatially regulate Z ring positioning in E. coli and are dynamically associated with the membrane. MinD binds to vesicles in the presence of ATP and can recruit MinC or MinE. Biochemical and genetic evidence indicate the binding sites for these two proteins on MinD overlap. Here we solved the structure of a hydrolytic-deficient mutant of MinD truncated for the C-terminal amphipathic helix involved in binding to the membrane. The structure solved in the presence of ATP is a dimer and reveals the face of MinD abutting the membrane. Using a combination of random and extensive site-directed mutagenesis additional residues important for MinE and MinC binding were identified. The location of these residues on the MinD structure confirms that the binding sites overlap and reveals that the binding sites are at the dimer interface and exposed to the cytosol. The location of the binding sites at the dimer interface offers a simple explanation for the ATP-dependency of MinC and MinE binding to MinD. PMID:21231967

Modification of opiate agonist binding by pertussis toxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abood, M.E.; Lee, N.M.; Loh, H.H.

1986-03-05

Opiate agonist binding is decreased by GTP, suggesting the possible involvement of GTP binding proteins in regulation of opiate receptor binding. This possibility was addressed by asking whether pertussis toxin treatment, which results in ADP-ribosylation and modification of G proteins, would alter opiate agonist binding. The striatum was chosen for the initial brain area to be studied, since regulation of opiate action in this area had been shown to be modified by pertussis toxin. Treatment of striatal membranes with pertussis toxin results in up to a 55% decrease in /sup 3/(H)-DADLE binding as compared with membranes treated identically without toxin.more » This corresponds to a near complete ADP-ribosylation of both G proteins in the striatal membrane. The decrease in agonist binding appears to be due to an altered affinity of the receptor for agonist as opposed to a decrease in the number of sites. This effect of pertussis toxin on opiate agonist binding demonstrates the actual involvement of G proteins in regulation of opiate receptor binding.« less

[125I]2-(2-chloro-4-iodo-phenylamino)-5-methyl-pyrroline (LNP 911), a high-affinity radioligand selective for I1 imidazoline receptors.

PubMed

Greney, Hugues; Urosevic, Dragan; Schann, Stephan; Dupuy, Laurence; Bruban, Véronique; Ehrhardt, Jean-Daniel; Bousquet, Pascal; Dontenwill, Monique

2002-07-01

The I1 subtype of imidazoline receptors (I1R) is a plasma membrane protein that is involved in diverse physiological functions. Available radioligands used so far to characterize the I(1)R were able to bind with similar affinities to alpha2-adrenergic receptors (alpha2-ARs) and to I1R. This feature was a major drawback for an adequate characterization of this receptor subtype. New imidazoline analogs were therefore synthesized and the present study describes one of these compounds, 2-(2-chloro-4-iodo-phenylamino)-5-methyl-pyrroline (LNP 911), which was of high affinity and selectivity for the I1R. LNP 911 was radioiodinated and its binding properties characterized in different membrane preparations. Saturation experiments with [125I]LNP 911 revealed a single high affinity binding site in PC-12 cell membranes (K(D) = 1.4 nM; B(max) = 398 fmol/mg protein) with low nonspecific binding. [125I]LNP 911 specific binding was inhibited by various imidazolines and analogs but was insensitive to guanosine-5'-O-(3-thio)triphosphate. The rank order of potency of some competing ligands [LNP 911, PIC, rilmenidine, 4-chloro-2-(imidazolin-2-ylamino)-isoindoline (BDF 6143), lofexidine, and clonidine] was consistent with the definition of [125I]LNP 911 binding sites as I1R. However, other high-affinity I1R ligands (moxonidine, efaroxan, and benazoline) exhibited low affinities for these binding sites in standard binding assays. In contrast, when [125I]LNP 911 was preincubated at 4 degrees C, competition curves of moxonidine became biphasic. In this case, moxonidine exhibited similar high affinities on [125I]LNP 911 binding sites as on I1R defined with [125I]PIC. Moxonidine proved also able to accelerate the dissociation of [125I]LNP 911 from its binding sites. These results suggest the existence of an allosteric modulation at the level of the I1R, which seems to be corroborated by the dose-dependent enhancement by LNP 911 of the agonist effects on the adenylate cyclase pathway associated to I1R. Because [125I]LNP 911 was unable to bind to the I2 binding site and alpha2AR, our data indicate that [125I]LNP 911 is the first highly selective radioiodinated probe for I1R with a nanomolar affinity. This new tool should facilitate the molecular characterization of the I1 imidazoline receptor.

Functional Role of the Interaction between Polysialic Acid and Myristoylated Alanine-rich C Kinase Substrate at the Plasma Membrane

PubMed Central

Theis, Thomas; Mishra, Bibhudatta; von der Ohe, Maren; Loers, Gabriele; Prondzynski, Maksymilian; Pless, Ole; Blackshear, Perry J.; Schachner, Melitta; Kleene, Ralf

2013-01-01

Polysialic acid (PSA) is a homopolymeric glycan that plays crucial roles in the developing and adult nervous system. So far only a few PSA-binding proteins have been identified. Here, we identify myristoylated alanine-rich C kinase substrate (MARCKS) as novel PSA binding partner. Binding assays showed a direct interaction between PSA and a peptide comprising the effector domain of MARCKS (MARCKS-ED). Co-immunoprecipitation of PSA-carrying neural cell adhesion molecule (PSA-NCAM) with MARCKS and co-immunostaining of MARCKS and PSA at the cell membrane of hippocampal neurons confirm the interaction between PSA and MARCKS. Co-localization and an intimate interaction of PSA and MARCKS at the cell surface was seen by confocal microscopy and fluorescence resonance energy transfer (FRET) analysis after the addition of fluorescently labeled PSA or PSA-NCAM to live CHO cells or hippocampal neurons expressing MARCKS as a fusion protein with green fluorescent protein (GFP). Cross-linking experiments showed that extracellularly applied PSA or PSA-NCAM and intracellularly expressed MARCKS-GFP are in close contact, suggesting that PSA and MARCKS interact with each other at the plasma membrane from opposite sides. Insertion of PSA and MARCKS-ED peptide into lipid bilayers from opposite sides alters the electric properties of the bilayer confirming the notion that PSA and the effector domain of MARCKS interact at and/or within the plane of the membrane. The MARCKS-ED peptide abolished PSA-induced enhancement of neurite outgrowth from cultured hippocampal neurons indicating an important functional role for the interaction between MARCKS and PSA in the developing and adult nervous system. PMID:23329829

Binding site stoichiometry and the effects of phosphorylation on human α1 homomeric glycine receptors

PubMed Central

Gentet, Luc J; Clements, John D

2002-01-01

The kinetic properties of the human α1 homomeric glycine receptor were investigated. Receptors were expressed in HEK 293 cells, and glycine was applied to outside-out membrane patches with sub-millisecond solution exchange. The activation time course of the glycine response was used to investigate receptor stoichiometry. The unbinding of three strychnine molecules and the cooperative binding of two glycine molecules were required to activate the channel. The effects of phosphorylation on glycine receptor kinetics were investigated by pretreating cells with phosphorylators or with phosphatases. Phosphorylation accelerated desensitisation, but slowed deactivation and recovery from desensitisation. A chemical-kinetic model was developed that reproduced the experimental observations. The model suggests that only three binding sites on the glycine channel are functional, while the remaining two binding sites are ‘silent’, possibly due to strong negative cooperativity. PMID:12356883

[(3)H]8-Ethyl-4-methyl-2-phenyl-(8R)-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]-purin-5-one ([(3)H]PSB-11), a novel high-affinity antagonist radioligand for human A(3) adenosine receptors.

PubMed

Müller, Christa E; Diekmann, Martina; Thorand, Mark; Ozola, Vita

2002-02-11

This study describes the preparation and binding properties of [(3)H]PSB-11, a novel, potent, and selective antagonist radioligand for human A(3) adenosine receptors (ARs). [(3)H]PSB-11 binding to membranes of Chinese hamster ovary (CHO) cells expressing the human A(3) AR was saturable and reversible. Saturation experiments showed that [(3)H]PSB-11 labeled a single class of binding sites with high affinity (K(D)=4.9 nM) and limited capacity (B(max)=3500 fmol/mg of protein). PSB-11 is highly selective versus the other adenosine receptor subtypes. The new radioligand shows an extraordinarily low degree of non-specific binding rendering it a very useful tool for studying the (patho)physiological roles of A(3 )ARs.

Glycoconjugate pattern of membranes in the acinar cell of the rat pancreas.

PubMed

Willemer, S; Köhler, H; Naumann, R; Kern, H F; Adler, G

1990-01-01

Lectin-binding studies were performed at the ultrastructural level to characterize glycoconjugate patterns on membrane systems in pancreatic acinar cells of the rat. Five lectins reacting with different sugar moieties were applied to ultrathin frozen sections: concanavalin A (ConA): glucose, mannose; wheat-germ agglutinin (WGA): N-acetylglucosamine, sialic acid; Ricinus communis agglutinin I (RCA I): galactose; Ulex europaeus agglutinin I (UEA I): L-fucose; soybean agglutinin (SBA): N-acetylgalactosamine). Binding sites of lectins were visualized either by direct conjugation to colloidal gold or by the use of a three-step procedure involving additional immune reactions. The rough endoplasmic reticulum and the nuclear envelope of acinar cells was selectively labelled for ConA. The membranes of the Golgi apparatus bound all lectins applied with an increasing intensity proceeding from the cis- to the trans-Golgi area for SBA, UEA I and WGA. In contrast RCA I selectively labelled the trans-Golgi cisternae. The membranes of condensing vacuoles and zymogen granules were labelled for all lectins used although the density of the label differed between the lectins. In contrast the content of zymogen granules failed to bind SBA and WGA. Lysosomal bodies (membranes and content) revealed binding sites for all lectins used. The plasma membranes were heavily labelled by all lectins except for SBA which showed only a weak binding to the lateral and the apical plasma membrane. These results are in accordance to current biochemical knowledge of the successive steps in the glycosylation of membrane proteins. It could be demonstrated, that the cryo-section technique is suitable for the fine structural localisation of surface glycoconjugates of plasma membranes and internal membranes in pancreatic acinar cells using plant lectins.

Modulation of receptor-mediated gonadotropin action in rat testes by dietary fat.

PubMed

Sebokova, E; Garg, M L; Clandinin, M T

1988-06-01

The effect of feeding diets enriched with 18:2 omega 6, 18:3 omega 3, or saturated fatty acids on lipid composition and receptor-mediated action of luteinizing hormone/human chorionic gonadotropin (LH/hCG) in rat testicular plasma membranes was investigated. Linoleic and alpha-linolenic acid treatments reduced total phospholipid and cholesterol content of the testicular plasma membrane and altered membrane phospholipid composition. Change in phospholipid and cholesterol content after feeding the polyunsaturated fats decreased cholesterol to phospholipid ratios and binding capacity of the LH/hCG receptor in the testicular plasma membrane. LH-stimulated adenylate cyclase activity was decreased in animals fed the linolenic acid-rich diet. NaF-stimulated adenylate cyclase activity was decreased in animals fed diets high in either polyunsaturated fatty acid. Decreased plasma membrane LH/hCG receptor content was associated with decreased testosterone production in Leydig cells in response to LH in the linolenic acid-fed group. It is suggested that change in cholesterol-to-phospholipid ratios alters the physical properties of testicular plasma membranes in a manner that influences accessibility of LH/hCG receptors in testicular tissue.

Detergent Optimized Membrane Protein Reconstitution in Liposomes for Solid State NMR

PubMed Central

2015-01-01

For small helical membrane proteins, their structures are highly sensitive to their environment, and solid state NMR is a structural technique that can characterize these membrane proteins in native-like lipid bilayers and proteoliposomes. To date, a systematic method by which to evaluate the effect of the solubilizing detergent on proteoliposome preparations for solid state NMR of membrane proteins has not been presented in the literature. A set of experiments are presented aimed at determining the conditions most amenable to dialysis mediated reconstitution sample preparation. A membrane protein from M. tuberculosis is used to illustrate the method. The results show that a detergent that stabilizes the most protein is not always ideal and sometimes cannot be removed by dialysis. By focusing on the lipid and protein binding properties of the detergent, proteoliposome preparations can be readily produced, which provide double the signal-to-noise ratios for both the oriented sample and magic angle spinning solid state NMR. The method will allow more membrane protein drug targets to be structurally characterized in lipid bilayer environments. PMID:24665863

Spring-Loaded Model Revisited: Paramyxovirus Fusion Requires Engagement of a Receptor Binding Protein beyond Initial Triggering of the Fusion Protein▿

PubMed Central

Porotto, Matteo; DeVito, Ilaria; Palmer, Samantha G.; Jurgens, Eric M.; Yee, Jia L.; Yokoyama, Christine C.; Pessi, Antonello; Moscona, Anne

2011-01-01

During paramyxovirus entry into a host cell, receptor engagement by a specialized binding protein triggers conformational changes in the adjacent fusion protein (F), leading to fusion between the viral and cell membranes. According to the existing paradigm of paramyxovirus membrane fusion, the initial activation of F by the receptor binding protein sets off a spring-loaded mechanism whereby the F protein progresses independently through the subsequent steps in the fusion process, ending in membrane merger. For human parainfluenza virus type 3 (HPIV3), the receptor binding protein (hemagglutinin-neuraminidase [HN]) has three functions: receptor binding, receptor cleaving, and activating F. We report that continuous receptor engagement by HN activates F to advance through the series of structural rearrangements required for fusion. In contrast to the prevailing model, the role of HN-receptor engagement in the fusion process is required beyond an initiating step, i.e., it is still required even after the insertion of the fusion peptide into the target cell membrane, enabling F to mediate membrane merger. We also report that for Nipah virus, whose receptor binding protein has no receptor-cleaving activity, the continuous stimulation of the F protein by a receptor-engaged binding protein is key for fusion. We suggest a general model for paramyxovirus fusion activation in which receptor engagement plays an active role in F activation, and the continued engagement of the receptor binding protein is essential to F protein function until the onset of membrane merger. This model has broad implications for the mechanism of paramyxovirus fusion and for strategies to prevent viral entry. PMID:21976650

Spring-loaded model revisited: paramyxovirus fusion requires engagement of a receptor binding protein beyond initial triggering of the fusion protein.

PubMed

Porotto, Matteo; Devito, Ilaria; Palmer, Samantha G; Jurgens, Eric M; Yee, Jia L; Yokoyama, Christine C; Pessi, Antonello; Moscona, Anne

2011-12-01

During paramyxovirus entry into a host cell, receptor engagement by a specialized binding protein triggers conformational changes in the adjacent fusion protein (F), leading to fusion between the viral and cell membranes. According to the existing paradigm of paramyxovirus membrane fusion, the initial activation of F by the receptor binding protein sets off a spring-loaded mechanism whereby the F protein progresses independently through the subsequent steps in the fusion process, ending in membrane merger. For human parainfluenza virus type 3 (HPIV3), the receptor binding protein (hemagglutinin-neuraminidase [HN]) has three functions: receptor binding, receptor cleaving, and activating F. We report that continuous receptor engagement by HN activates F to advance through the series of structural rearrangements required for fusion. In contrast to the prevailing model, the role of HN-receptor engagement in the fusion process is required beyond an initiating step, i.e., it is still required even after the insertion of the fusion peptide into the target cell membrane, enabling F to mediate membrane merger. We also report that for Nipah virus, whose receptor binding protein has no receptor-cleaving activity, the continuous stimulation of the F protein by a receptor-engaged binding protein is key for fusion. We suggest a general model for paramyxovirus fusion activation in which receptor engagement plays an active role in F activation, and the continued engagement of the receptor binding protein is essential to F protein function until the onset of membrane merger. This model has broad implications for the mechanism of paramyxovirus fusion and for strategies to prevent viral entry.

Fluorescence studies on the interaction of choline-binding domain B of the major bovine seminal plasma protein, PDC-109 with phospholipid membranes.

PubMed

Damai, Rajani S; Anbazhagan, V; Rao, K Babu; Swamy, Musti J

2009-12-01

The microenvironment and accessibility of the tryptophan residues in domain B of PDC-109 (PDC-109/B) in the native state and upon ligand binding have been investigated by fluorescence quenching, time-resolved fluorescence and red-edge excitation shift (REES) studies. The increase in the intrinsic fluorescence emission intensity of PDC-109/B upon binding to lysophosphatidylcholine (Lyso-PC) micelles and dimyristoylphosphatidylcholine (DMPC) membranes was considerably less as compared to that observed with the whole PDC-109 protein. The degree of quenching achieved by different quenchers with PDC-109/B bound to Lyso-PC and DMPC membranes was significantly higher as compared to the full PDC-109 protein, indicating that membrane binding afforded considerably lesser protection to the tryptophan residues of domain B as compared to those in the full PDC-109 protein. Finally, changes in red-edge excitation shift (REES) seen with PDC-109/B upon binding to DMPC membranes and Lyso-PC micelles were smaller that the corresponding changes in the REES values observed for the full PDC-109. These results, taken together suggest that intact PDC-109 penetrates deeper into the hydrophobic parts of the membrane as compared to domain B alone, which could be the reason for the inability of PDC-109/B to induce cholesterol efflux, despite its ability to recognize choline phospholipids at the membrane surface.

Functional domains of the T lymphocyte plasma membrane: characterization of the polypeptide composition.

PubMed

Szamel, M; Kaever, V; Resch, K

1987-01-01

Highly purified plasma membranes from calf thymocytes were fractionated by affinity chromatography on Concanavalin A-Sepharose into two subfractions, one eluting freely from the affinity column (MF1) and a second being specifically retained (MF2). SDS-polyacrylamide-gel-electrophoresis revealed different polypeptide patterns of the two plasma membrane subfractions. Polypeptides of apparent molecular weights of 170, 150, 110, 94, 39, and 30 kDa were several-fold enriched in the adherent fraction, MF2. In contrast, several proteins in the 55-65 kDa range were preferentially recovered in the non-adherent fraction. Five Five of the six polypeptides, preferentially recovered in MF2 proved to be glycoproteins, the 39 kDa peptide was non-glycosilated. The differences in the amounts of the polypeptides specifically enriched in the adherent fraction MF2 became even more clear-cut when plasma membranes solubilized with non-ionic detergents (lysolecithin, ET-18-2H, Triton-X-100) were separated by affinity chromatography on Concanavalin A-Sepharose. The non-glycosilated peptide of apparent molecular weight of 39 kDa was recovered together with several glycoproteins in the adherent fraction, MF2, suggesting that not single glycoproteins, but plasma membrane domains were separated by Concanavalin A-Sepharose. Although the glycoproteins of the non-adherent fraction MF1 bound significant amounts of Concanavalin A, the major Concanavalin A binding glycoproteins were recovered in the adherent fraction, MF2. The plasma membrane subfractions showed also different functional properties, the specific activities [Na+ + K+]AT-Pase, Ca2+ ATPase and lysolecithin acyltransferase were several-fold enriched in the adherent fraction, MF2, as compared to MF1. The data suggest the existence of plasma membrane domains in the plasma membranes of thymocytes consisting of a different set of proteins, among others the major Concanavalin A binding glycoproteins with some membrane bound enzymes, probably implicated in the initiation of lymphocyte activation.

Structural features and lipid binding domain of tubulin on biomimetic mitochondrial membranes

PubMed Central

Hoogerheide, David P.; Noskov, Sergei Y.; Jacobs, Daniel; Bergdoll, Lucie; Silin, Vitalii; Worcester, David L.; Abramson, Jeff; Nanda, Hirsh; Rostovtseva, Tatiana K.; Bezrukov, Sergey M.

2017-01-01

Dimeric tubulin, an abundant water-soluble cytosolic protein known primarily for its role in the cytoskeleton, is routinely found to be associated with mitochondrial outer membranes, although the structure and physiological role of mitochondria-bound tubulin are still unknown. There is also no consensus on whether tubulin is a peripheral membrane protein or is integrated into the outer mitochondrial membrane. Here the results of five independent techniques—surface plasmon resonance, electrochemical impedance spectroscopy, bilayer overtone analysis, neutron reflectometry, and molecular dynamics simulations—suggest that α-tubulin’s amphipathic helix H10 is responsible for peripheral binding of dimeric tubulin to biomimetic “mitochondrial” membranes in a manner that differentiates between the two primary lipid headgroups found in mitochondrial membranes, phosphatidylethanolamine and phosphatidylcholine. The identification of the tubulin dimer orientation and membrane-binding domain represents an essential step toward our understanding of the complex mechanisms by which tubulin interacts with integral proteins of the mitochondrial outer membrane and is important for the structure-inspired design of tubulin-targeting agents. PMID:28420794

Pressure modulation of Ras-membrane interactions and intervesicle transfer.

PubMed

Kapoor, Shobhna; Werkmüller, Alexander; Goody, Roger S; Waldmann, Herbert; Winter, Roland

2013-04-24

Proteins attached to the plasma membrane frequently encounter mechanical stresses, including high hydrostatic pressure (HHP) stress. Signaling pathways involving membrane-associated small GTPases (e.g., Ras) have been identified as critical loci for pressure perturbation. However, the impact of mechanical stimuli on biological outputs is still largely terra incognita. The present study explores the effect of HHP on the membrane association, dissociation, and intervesicle transfer process of N-Ras by using a FRET-based assay to obtain the kinetic parameters and volumetric properties along the reaction path of these processes. Notably, membrane association is fostered upon pressurization. Conversely, depending on the nature and lateral organization of the lipid membrane, acceleration or retardation is observed for the dissociation step. In addition, HHP can be inferred as a positive regulator of N-Ras clustering, in particular in heterogeneous membranes. The susceptibility of membrane interaction to pressure raises the idea of a role of lipidated signaling molecules as mechanosensors, transducing mechanical stimuli to chemical signals by regulating their membrane binding and dissociation. Finally, our results provide first insights into the influence of pressure on membrane-associated Ras-controlled signaling events in organisms living under extreme environmental conditions such as those that are encountered in the deep sea and sub-seafloor environments, where pressures reach the kilobar (100 MPa) range.

Modulation of GABAergic receptor binding by activation of calcium and calmodulin-dependent kinase II membrane phosphorylation.

PubMed

Churn, S B; DeLorenzo, R J

1998-10-26

gamma-Aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the central nervous system (CNS). Because of the important role that GABA plays in the CNS, alteration of GABAA receptor function would significantly affect neuronal excitability. Protein phosphorylation is a major mechanism for regulating receptor function in the brain and has been implicated in modulating GABAA receptor function. Therefore, this study was initiated to determine the role of calmodulin-dependent kinase II (CaM kinase II) membrane phosphorylation on GABAA receptor binding. Synaptosomal membrane fractions were tested for CaM kinase II activity towards endogenous substrates. In addition, muscimol binding was evaluated under equilibrium conditions in synaptosomal membrane fractions subjected to either basal (Mg2+ alone) or maximal CaM kinase II-dependent phosphorylation. Activation of endogenous CaM kinase II-dependent phosphorylation resulted in a significant enhancement of the apparent Bmax for muscimol binding without significantly altering the apparent binding affinity. The enhanced muscimol binding could be increased further by the addition of exogenous CaM kinase II to synaptosomal membrane fractions. Co-incubation with inhibitors of kinase activity during the phosphorylation reactions blocked the CaM kinase II-dependent increase in muscimol binding. The data support the hypothesis that activation of CaM kinase II-dependent phosphorylation caused an increased GABAA receptor binding and may play an important role in modulating the function of this inhibitory receptor/chloride ion channel complex. Copyright 1998 Elsevier Science B.V.

The hepta-beta-glucoside elicitor-binding proteins from legumes represent a putative receptor family.

PubMed

Mithöfer, A; Fliegmann, J; Neuhaus-Url, G; Schwarz, H; Ebel, J

2000-08-01

The ability of legumes to recognize and respond to beta-glucan elicitors by synthesizing phytoalexins is consistent with the existence of a membrane-bound beta-glucan-binding site. Related proteins of approximately 75 kDa and the corresponding mRNAs were detected in various species of legumes which respond to beta-glucans. The cDNAs for the beta-glucan-binding proteins of bean and soybean were cloned. The deduced 75-kDa proteins are predominantly hydrophilic and constitute a unique class of glucan-binding proteins with no currently recognizable functional domains. Heterologous expression of the soybean beta-glucan-binding protein in tomato cells resulted in the generation of a high-affinity binding site for the elicitor-active hepta-beta-glucoside conjugate (Kd = 4.5 nM). Ligand competition experiments with the recombinant binding sites demonstrated similar ligand specificities when compared with soybean. In both soybean and transgenic tomato, membrane-bound, active forms of the glucan-binding proteins coexist with immunologically detectable, soluble but inactive forms of the proteins. Reconstitution of a soluble protein fraction into lipid vesicles regained beta-glucoside-binding activity but with lower affinity (Kd = 130 nM). We conclude that the beta-glucan elicitor receptors of legumes are composed of the 75 kDa glucan-binding proteins as the critical components for ligand-recognition, and of an as yet unknown membrane anchor constituting the plasma membrane-associated receptor complex.

Clostridium Perfringens Epsilon Toxin Binds to Membrane Lipids and Its Cytotoxic Action Depends on Sulfatide.

PubMed

Gil, Carles; Dorca-Arévalo, Jonatan; Blasi, Juan

2015-01-01

Epsilon toxin (Etx) is one of the major lethal toxins produced by Clostridium perfringens types B and D, being the causal agent of fatal enterotoxemia in animals, mainly sheep and goats. Etx is synthesized as a non-active prototoxin form (proEtx) that becomes active upon proteolytic activation. Etx exhibits a cytotoxic effect through the formation of a pore in the plasma membrane of selected cell targets where Etx specifically binds due to the presence of specific receptors. However, the identity and nature of host receptors of Etx remain a matter of controversy. In the present study, the interactions between Etx and membrane lipids from the synaptosome-enriched fraction from rat brain (P2 fraction) and MDCK cell plasma membrane preparations were analyzed. Our findings show that both Etx and proEtx bind to lipids extracted from lipid rafts from the two different models as assessed by protein-lipid overlay assay. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. Binding of proEtx to sulfatide, phosphatidylserine, phosphatidylinositol (3)-phosphate and phosphatidylinositol (5)-phosphate was detected. Removal of the sulphate groups via sulfatase treatment led to a dramatic decrease in Etx-induced cytotoxicity, but not in proEtx-GFP binding to MDCK cells or a significant shift in oligomer formation, pointing to a role of sulfatide in pore formation in rafts but not in toxin binding to the target cell membrane. These results show for the first time the interaction between Etx and membrane lipids from host tissue and point to a major role for sulfatides in C. perfringens epsilon toxin pathophysiology.

Human Atg8-cardiolipin interactions in mitophagy: Specific properties of LC3B, GABARAPL2 and GABARAP.

PubMed

Antón, Zuriñe; Landajuela, Ane; Hervás, Javier H; Montes, L Ruth; Hernández-Tiedra, Sonia; Velasco, Guillermo; Goñi, Felix M; Alonso, Alicia

2016-12-01

The phospholipid cardiolipin (CL) has been proposed to play a role in selective mitochondrial autophagy, or mitophagy. CL externalization to the outer mitochondrial membrane would act as a signal for the human Atg8 ortholog subfamily, MAP1LC3 (LC3). The latter would mediate both mitochondrial recognition and autophagosome formation, ultimately leading to removal of damaged mitochondria. We have applied quantitative biophysical techniques to the study of CL interaction with various Atg8 human orthologs, namely LC3B, GABARAPL2 and GABARAP. We have found that LC3B interacts preferentially with CL over other di-anionic lipids, that CL-LC3B binding occurs with positive cooperativity, and that the CL-LC3B interaction relies only partially on electrostatic forces. CL-induced increased membrane fluidity appears also as an important factor helping LC3B to bind CL. The LC3B C terminus remains exposed to the hydrophilic environment after protein binding to CL-enriched membranes. In intact U87MG human glioblastoma cells rotenone-induced autophagy leads to LC3B translocation to mitochondria and subsequent delivery of mitochondria to lysosomes. We have also observed that GABARAP, but not GABARAPL2, interacts with CL in vitro. However neither GABARAP nor GABARAPL2 were translocated to mitochondria in rotenone-treated U87MG cells. Thus the various human Atg8 orthologs might play specific roles in different autophagic processes.

N-Lauroylation during the Expression of Recombinant N-Myristoylated Proteins: Implications and Solutions.

PubMed

Flamm, Andrea Gabriele; Le Roux, Anabel-Lise; Mateos, Borja; Díaz-Lobo, Mireia; Storch, Barbara; Breuker, Kathrin; Konrat, Robert; Pons, Miquel; Coudevylle, Nicolas

2016-01-01

Incorporation of myristic acid onto the N terminus of a protein is a crucial modification that promotes membrane binding and correct localization of important components of signaling pathways. Recombinant expression of N-myristoylated proteins in Escherichia coli can be achieved by co-expressing yeast N-myristoyltransferase and supplementing the growth medium with myristic acid. However, undesired incorporation of the 12-carbon fatty acid lauric acid can also occur (leading to heterogeneous samples), especially when the available carbon sources are scarce, as it is the case in minimal medium for the expression of isotopically enriched samples. By applying this method to the brain acid soluble protein 1 and the 1-185 N-terminal region of c-Src, we show the significant, and protein-specific, differences in the membrane binding properties of lauroylated and myristoylated forms. We also present a robust strategy for obtaining lauryl-free samples of myristoylated proteins in both rich and minimal media. © 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Bacillus thuringiensis Cry1A toxin-binding glycoconjugates present on the brush border membrane and in the peritrophic membrane of the Douglas-fir tussock moth are peritrophins

Treesearch

Algimantas P. Valaitis; John D. Podgwaite

2013-01-01

Bacillus thuringiensis (Bt) Cry1A toxin-binding sites in the Douglas fir tussock moth (DFTM) larval gut were localized using immunofluorescence microscopy. Cry1Aa, Cry1Ab and Cry1Ac all bound strongly to the DFTM peritrophic membrane (PM); weaker binding of the Cry1A toxins was observed along the apical brush border of the midgut epithelium....

Analysis of the interaction between membrane proteins and soluble binding partners by surface plasmon resonance.

PubMed

Wu, Zht Cheng; de Keyzer, Jeanine; Kusters, Ilja; Driessen, Arnold J M

2013-01-01

The interaction between membrane proteins and their (protein) ligands is conventionally investigated by nonequilibrium methods such as co-sedimentation or pull-down assays. Surface Plasmon Resonance can be used to monitor such binding events in real-time using isolated membranes immobilized to a surface providing insights in the kinetics of binding under equilibrium conditions. This application provides a fast, automated way to detect interacting species and to determine the kinetics and affinity (Kd) of the interaction.

Part I: an x-ray scattering study of cholera toxin penetration and induced phase transformations in lipid membranes.

PubMed

Miller, C E; Majewski, J; Watkins, E B; Kuhl, T L

2008-07-01

Cholera toxin is a highly efficient biotoxin, which is frequently used as a tool to investigate protein-membrane interactions and as a reporter for membrane rafts. Cholera toxin binds selectively to gangliosides with highest affinity to GM(1). However, the mechanism by which cholera toxin crosses the membrane remains unresolved. Using x-ray reflectivity and grazing incidence diffraction, we have been able to monitor the binding and penetration of cholera toxin into a model lipid monolayer containing the receptor GM(1) at the air-water interface. Very high toxin coverage was obtained allowing precise measurements of how toxin binding alters lipid packing. Grazing incidence x-ray diffraction revealed the coexistence of two monolayer phases after toxin binding. The first was identical to the monolayer before toxin binding. In regions where toxin was bound, a second membrane phase exhibited a decrease in order as evidenced by a larger area per molecule and tilt angle with concomitant thinning of the monolayer. These results demonstrate that cholera toxin binding induces the formation of structurally distinct, less ordered domains in gel phases. Furthermore, the largest decrease in lateral order to the monolayer occurred at low pH, supporting a low endosomal pH in the infection pathway. Surprisingly, at pH = 8 toxin penetration by the binding portion of the toxin, the B(5) pentamer, was also observed.

Atomistic models for free energy evaluation of drug binding to membrane proteins.

PubMed

Durdagi, S; Zhao, C; Cuervo, J E; Noskov, S Y

2011-01-01

The binding of various molecules to integral membrane proteins with optimal affinity and specificity is central to normal function of cell. While membrane proteins represent about one third of the whole cell proteome, they are a majority of common drug targets. The quest for the development of computational models capable of accurate evaluation of binding affinities, decomposition of the binding into its principal components and thus mapping molecular mechanisms of binding remains one of the main goals of modern computational biophysics and related drug development. The primary scope of this review will be on the recent extension of computational methods for the study of drug binding to membrane proteins. Several examples of such applications will be provided ranging from secondary transporters to voltage gated channels. In this mini-review, we will provide a short summary on the breadth of different methods for binding affinity evaluation. These methods include molecular docking with docking scoring functions, molecular dynamics (MD) simulations combined with post-processing analysis using Molecular Mechanics/Poisson Boltzmann (Generalized Born) Surface Area (MM/PB(GB)SA), as well as direct evaluation of free energies from Free Energy Perturbation (FEP) with constraining schemes, and Potential of Mean Force (PMF) computations. We will compare advantages and shortcomings of popular techniques and provide discussion on the integrative strategies for drug development aimed at targeting membrane proteins.

Large enhancement of functional activity of active site-inhibited factor VIIa due to protein dimerization: insights into mechanism of assembly/disassembly from tissue factor.

PubMed

Stone, Matthew D; Harvey, Stephen B; Martinez, Michael B; Bach, Ronald R; Nelsestuen, Gary L

2005-04-26

Active site-inhibited blood clotting factor VIIa (fVIIai) binds to tissue factor (TF), a cell surface receptor that is exposed upon injury and initiates the blood clotting cascade. FVIIai blocks binding of the corresponding enzyme (fVIIa) or zymogen (fVII) forms of factor VII and inhibits coagulation. Although several studies have suggested that fVIIai may have superior anticoagulation effects in vivo, a challenge for use of fVIIai is cost of production. This study reports the properties of dimeric forms of fVIIai that are cross-linked through their active sites. Dimeric wild-type fVIIai was at least 75-fold more effective than monomeric fVIIai in blocking fVIIa association with TF. The dimer of a mutant fVIIai with higher membrane affinity was 1600-fold more effective. Anticoagulation by any form of fVIIai differed substantially from agents such as heparin and showed a delayed mode of action. Coagulation proceeded normally for the first minutes, and inhibition increased as equilibrium binding was established. It is suggested that association of fVIIa(i) with TF in a collision-dependent reaction gives equal access of inhibitor and enzyme to TF. Assembly was not influenced by the higher affinity and slower dissociation of the dimer. As a result, anticoagulation was delayed until the reaction reached equilibrium. Properties of different dissociation experiments suggested that dissociation of fVIIai from TF occurred by a two-step mechanism. The first step was separation of TF-fVIIa(i) while both proteins remained bound to the membrane, and the second step was dissociation of the fVIIa(i) from the membrane. These results suggest novel actions of fVIIai that distinguish it from most of the anticoagulants that block later steps of the coagulation cascade.

Vitreoscilla hemoglobin. Intracellular localization and binding to membranes.

PubMed

Ramandeep; Hwang, K W; Raje, M; Kim, K J; Stark, B C; Dikshit, K L; Webster, D A

2001-07-06

The obligate aerobic bacterium, Vitreoscilla, synthesizes elevated quantities of a homodimeric hemoglobin (VHb) under hypoxic growth conditions. Expression of VHb in heterologous hosts often enhances growth and product formation. A role in facilitating oxygen transfer to the respiratory membranes is one explanation of its cellular function. Immunogold labeling of VHb in both Vitreoscilla and recombinant Escherichia coli bearing the VHb gene clearly indicated that VHb has a cytoplasmic (not periplasmic) localization and is concentrated near the periphery of the cytosolic face of the cell membrane. OmpA signal-peptide VHb fusions were transported into the periplasm in E. coli, but this did not confer any additional growth advantage. The interaction of VHb with respiratory membranes was also studied. The K(d) values for the binding of VHb to Vitreoscilla and E. coli cell membranes were approximately 5-6 microm, a 4-8-fold higher affinity than those of horse myoglobin and hemoglobin for these same membranes. VHb stimulated the ubiquinol-1 oxidase activity of inverted Vitreoscilla membranes by 68%. The inclusion of Vitreoscilla cytochrome bo in proteoliposomes led to 2.4- and 6-fold increases in VHb binding affinity and binding site number, respectively, relative to control liposomes, suggesting a direct interaction between VHb and cytochrome bo.

High-resolution Structures of Protein-Membrane Complexes by Neutron Reflection and MD Simulation: Membrane Association of the PTEN Tumor Suppressor

NASA Astrophysics Data System (ADS)

Lösche, Matthias

2012-02-01

The lipid matrix of biomembranes is an in-plane fluid, thermally and compositionally disordered leaflet of 5 nm thickness and notoriously difficult to characterize in structural terms. Yet, biomembranes are ubiquitous in the cell, and membrane-bound proteins are implicated in a variety of signaling pathways and intra-cellular transport. We developed methodology to study proteins associated with model membranes using neutron reflection measurements and showed recently that this approach can resolve the penetration depth and orientation of membrane proteins with ångstrom resolution if their crystal or NMR structure is known. Here we apply this technology to determine the membrane bindung and unravel functional details of the PTEN phosphatase, a key player in the PI3K apoptosis pathway. PTEN is an important regulatory protein and tumor suppressor that performs its phosphatase activity as an interfacial enzyme at the plasma membrane-cytoplasm boundary. Acting as an antagonist to phosphoinositide-3-kinase (PI3K) in cell signaling, it is deleted in many human cancers. Despite its importance in regulating the levels of the phosphoinositoltriphosphate PI(3,4,5)P3, there is little understanding of how PTEN binds to membranes, is activated and then acts as a phosphatase. We investigated the structure and function of PTEN by studying its membrane affinity and localization on in-plane fluid, thermally disordered synthetic membrane models. The membrane association of the protein depends strongly on membrane composition, where phosphatidylserine (PS) and phosphatidylinositol diphosphate (PI(4,5)P2) act synergetically in attracting the enzyme to the membrane surface. Membrane affinities depend strongly on membrane fluidity, which suggests multiple binding sites on the protein for PI(4,5)P2. Neutron reflection measurements show that the PTEN phosphatase ``scoots'' along the membrane surface (penetration < 5 å) but binds the membrane tightly with its two major domains, the C2 and phosphatase domains. In the bound state, PTEN's regulatory C-terminal tail is displaced from the membrane and organized on the far side of the protein, ˜ 60 å away from the bilayer surface, in a rather compact structure. The combination of binding studies and neutron reflection allows us to distinguish between PTEN mutant proteins and ultimately may identify the structural features required for membrane binding and activation of PTEN. Molecular dynamics simulations, currently in progress, refine this structural picture further.

Electrostatic interactions and binding orientation of HIV-1 matrix studied by neutron reflectivity.

PubMed

Nanda, Hirsh; Datta, Siddhartha A K; Heinrich, Frank; Lösche, Mathias; Rein, Alan; Krueger, Susan; Curtis, Joseph E

2010-10-20

The N-terminal matrix (MA) domain of the HIV-1 Gag protein is responsible for binding to the plasma membrane of host cells during viral assembly. The putative membrane-binding interface of MA was previously mapped by means of mutagenesis and analysis of its trimeric crystal structure. However, the orientation of MA on membranes has not been directly determined by experimental measurements. We present neutron reflectivity measurements that resolve the one-dimensional scattering length density profile of MA bound to a biomimetic of the native viral membrane. A molecular refinement procedure was developed using atomic structures of MA to determine the orientation of the protein on the membrane. The orientation defines a lipid-binding interface consistent with previous mutagenesis results. The MA protein maintains this orientation without the presence of a myristate group, driven only by electrostatic interactions. Furthermore, MA is found to penetrate the membrane headgroup region peripherally such that only the side chains of specific Lys and Arg residues interact with the surface. The results suggest that electrostatic interactions are sufficient to favorably orient MA on viral membrane mimics. The spatial determination of the membrane-bound protein demonstrates the ability of neutron reflectivity to discern orientation and penetration under physiologically relevant conditions. Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Involvement of the N-terminal region in alpha-crystallin-lens membrane recognition

NASA Technical Reports Server (NTRS)

Ifeanyi, F.; Takemoto, L.; Spooner, B. S. (Principal Investigator)

1991-01-01

Previous studies have demonstrated that alpha-crystallin binds specifically, in a saturable manner, to lens membrane. To determine the region of the alpha-crystallin molecule that might be involved in this binding, native alpha-crystallin from the bovine lens has been treated by limited digestion with trypsin, to produce alpha-A molecules with an intact C-terminal region, and a nicked N-terminal region. Compared to intact alpha-crystallin, trypsin-treated alpha-crystallin binds less avidly to lens membrane, suggesting that the N-terminal region of the alpha-A molecule may play a key role in the recognition between lens membrane and crystallin.

Binding Affinity Effects on Physical Characteristics of a Model Phase-Separated Protein Droplet

NASA Astrophysics Data System (ADS)

Chuang, Sara; Banani, Salman; Rosen, Michael; Brangwynne, Clifford

2015-03-01

Non-membrane bound organelles are associated with a range of biological functions. Several of these structures exhibit liquid-like properties, and may represent droplets of phase-separated RNA and/or proteins. These structures are often enriched in multi-valent molecules, however little is known about the interactions driving the assembly, properties, and function. Here, we address this question using a model multi-valent protein system consisting of repeats of Small Ubiquitin-like Modifier (SUMO) protein and a SUMO-interacting motif (SIM). These proteins undergo phase separation into liquid-like droplets. We combine microrheology and quantitative microscopy to determine affect of binding affinity on the viscosity, density and surface tension of these droplets. We also use fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and partitioning experiments to probe the structure and dynamics within these droplets. Our results shed light on how inter-molecular interactions manifests in droplet properties, and lay the groundwork for a comprehensive biophysical picture of intracellular RNA/protein organelles.

Influence of freeze-thawing on hyaluronic acid binding of human spermatozoa.

PubMed

Nijs, Martine; Creemers, Eva; Cox, Annemie; Janssen, Mia; Vanheusden, Elke; Castro-Sanchez, Yovanna; Thijs, Herbert; Ombelet, Willem

2009-08-01

Mature human spermatozoa have at least three specific hyaluronic acid (HA) binding proteins present on their sperm membrane. These receptors play a role in the acrosome reaction, hyaluronidase activity, hyaluronan-mediated motility and sperm-zona and sperm-oolemmal binding. Cryopreservation of spermatozoa can cause ultrastructural and even molecular damage. The aim of this study was to investigate if HA binding receptors of human spermatozoa remain functional after freeze-thawing. Forty patients were enrolled in the study. Semen samples were analysed before and after cryopreservation. Parameters analysed included concentration, motility, morphology and hyaluronan binding. Samples were frozen in CBS straws using a glycerol-glucose-based cryoprotectant. HA binding was studied using the sperm-hyaluronan binding assay. Freeze-thawing resulted in a significant decline in motility: the percentage of motile spermatozoa reduced from 50.6 to 30.3% (P < 0.001). HA binding properties of frozen-thawed spermatozoa remained unchanged after the freeze-thawing process: 68.5 +/- 17.1% spermatozoa of the neat sample were bound to HA, as were 71.3 +/- 20.4 of the frozen-thawed sample. This study indicates that freeze-thawing did not alter the functional hyaluronan binding sites of mature motile spermatozoa, and therefore will not alter their fertilizing potential.

External location of sites on pig erythrocyte membranes that bind nitrobenzylthioinosine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Agbanyo, F.R.; Cass, C.E.; Paterson, A.R.

1988-03-01

Nucleoside transport in erythrocytes of various species is inhibited by the binding of nitrobenzylthioinosine (NBMPR) to high affinity sites associated with nucleoside transport elements of the plasma membrane. The present study examined binding of (/sup 3/H)NBMPR to unsealed ghosts and to sealed right-side-out vesicles (ROVs) and inside-out vesicles (IOVs) prepared from pig erythrocytes. Kd values for NBMPR dissociation from the ligand-site complex in unsealed ghosts, ROVs and IOVs were similar (1.6-2.4 nM), and Bmax values (mean +/- SD) were, respectively, 22.2 +/- 5.5, 25.8 +/- 6.4, and 37.3 +/- 4.0 molecules/fg of protein, reflecting differences in the protein content ofmore » the membrane preparations. When temperatures were decreased from 22 degrees to 4 degrees, NBMPR binding to erythrocyte membrane preparations was reduced in IOVs relative to that in unsealed ghosts and ROVs. At 22 degrees, the association of NBMPR molecules with IOVs was slower than with ROVs and unsealed ghosts, differences that were virtually eliminated by permeabilization of the membrane preparations with saponin. Thus, the binding sites were more accessible to external NBMPR in sealed ROVs and unsealed ghosts than in sealed IOVs, indicating that the NBMPR sites are located on the extracellular aspect of the membrane.« less

The HOPS/Class C Vps Complex Tethers High-Curvature Membranes via a Direct Protein-Membrane Interaction.

PubMed

Ho, Ruoya; Stroupe, Christopher

2016-10-01

Membrane tethering is a physical association of two membranes before their fusion. Many membrane tethering factors have been identified, but the interactions that mediate inter-membrane associations remain largely a matter of conjecture. Previously, we reported that the homotypic fusion and protein sorting/Class C vacuolar protein sorting (HOPS/Class C Vps) complex, which has two binding sites for the yeast vacuolar Rab GTPase Ypt7p, can tether two low-curvature liposomes when both membranes bear Ypt7p. Here, we show that HOPS tethers highly curved liposomes to Ypt7p-bearing low-curvature liposomes even when the high-curvature liposomes are protein-free. Phosphorylation of the curvature-sensing amphipathic lipid-packing sensor (ALPS) motif from the Vps41p HOPS subunit abrogates tethering of high-curvature liposomes. A HOPS complex without its Vps39p subunit, which contains one of the Ypt7p binding sites in HOPS, lacks tethering activity, though it binds high-curvature liposomes and Ypt7p-bearing low-curvature liposomes. Thus, HOPS tethers highly curved membranes via a direct protein-membrane interaction. Such high-curvature membranes are found at the sites of vacuole tethering and fusion. There, vacuole membranes bend sharply, generating large areas of vacuole-vacuole contact. We propose that HOPS localizes via the Vps41p ALPS motif to these high-curvature regions. There, HOPS binds via Vps39p to Ypt7p in an apposed vacuole membrane. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Fluorescent Probe Study of AOT Vesicle Membranes and Their Alteration upon Addition of Aniline or the Aniline Dimer p-Aminodiphenylamine (PADPA).

PubMed

Iwasaki, Fumihiko; Luginbühl, Sandra; Suga, Keishi; Walde, Peter; Umakoshi, Hiroshi

2017-02-28

Artificial vesicles formed from sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in aqueous solution are used successfully as additives for enzymatic oligomerizations or polymerizations of aniline or the aniline dimer p-aminodiphenylamine (PADPA) under slightly acidic conditions (e.g., pH 4.3 with horseradish peroxidase and hydrogen peroxide as oxidants). In these systems, the reactions occur membrane surface-confined. Therefore, (i) the physicochemical properties of the vesicle membrane and (ii) the interaction of aniline or PADPA with the AOT membrane play crucial roles in the progress and final outcome of the reactions. For this reason, the properties of AOT vesicles with and without added aniline or PADPA were investigated by using two fluorescent membrane probes: 1,6-diphenyl-1,3,5-hexatriene (DPH) and 6-lauroyl-2-dimethylaminonaphthalene (Laurdan). DPH and Laurdan were used as "sensors" of the membrane fluidity, surface polarity, and membrane phase state. Moreover, the effect of hexanol, alone or in combination with aniline or PADPA, as a possible modifier of the AOT membrane, was also studied with the aim of evaluating whether the membrane fluidity and surface polarity is altered significantly by hexanol, which, in turn, may have an influence on the mentioned types of reactions. The data obtained indicate that the AOT vesicle membrane at room temperature and pH 4.3 (0.1 M NaH 2 PO 4 ) is more fluid and has a more polar surface than in the case of fluid phospholipid vesicle membranes formed from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). Furthermore, the fluorescence measurements indicate that mixed AOT-hexanol membranes are less fluid than pure AOT membranes and that they have a lower surface polarity than pure AOT membranes. PADPA strongly binds to AOT and to mixed AOT/hexanol membranes and leads to drastic changes in the membrane properties (decrease in fluidity and surface polarity), resulting in Laurdan fluorescence spectra, which are characteristic for intramembrane phase separations (coexistence of ordered and disordered domains). This means that highly fluid AOT membranes transform upon the addition of PADPA into membranes that have ordered domains. Although the relevance of this finding for the enzymatic oligomerization of PADPA is not yet clear, it is also of interest if one likes to use heterogeneous vesicle membranes as additives for carrying out membrane surface-confined reactions that do not necessarily involve PADPA as a reactant.

Competitive Binding Assay for the G-Protein-Coupled Receptor 30 (GPR30) or G-Protein-Coupled Estrogen Receptor (GPER).

PubMed

Thekkumkara, Thomas; Snyder, Russell; Karamyan, Vardan T

2016-01-01

The role of 2-methoxyestradiol is becoming a major area of investigation because of its therapeutic utility, though its mechanism is not fully explored. Recent studies have identified the G-protein-coupled receptor 30 (GPR30, GPER) as a high-affinity membrane receptor for 2-methoxyestradiol. However, studies aimed at establishing the binding affinities of steroid compounds for specific targets are difficult, as the tracers are highly lipophilic and often result in nonspecific binding in lipid-rich membrane preparations with low-level target receptor expression. 2-Methoxyestradiol binding studies are essential to elucidate the underlying effects of this novel estrogen metabolite and to validate its targets; therefore, this competitive receptor-binding assay protocol was developed in order to assess the membrane receptor binding and affinity of 2-methyoxyestradiol.

Membrane Curvature Sensing by Amphipathic Helices

PubMed Central

Jensen, Martin Borch; Bhatia, Vikram Kjøller; Jao, Christine C.; Rasmussen, Jakob Ewald; Pedersen, Søren L.; Jensen, Knud J.; Langen, Ralf; Stamou, Dimitrios

2011-01-01

Preferential binding of proteins on curved membranes (membrane curvature sensing) is increasingly emerging as a general mechanism whereby cells may effect protein localization and trafficking. Here we use a novel single liposome fluorescence microscopy assay to examine a common sensing motif, the amphipathic helix (AH), and provide quantitative measures describing and distinguishing membrane binding and sensing behavior. By studying two AH-containing proteins, α-synuclein and annexin B12, as well as a range of AH peptide mutants, we reveal that both the hydrophobic and hydrophilic faces of the helix greatly influence binding and sensing. Although increased hydrophobic and electrostatic interactions with the membrane both lead to greater densities of bound protein, the former yields membrane curvature-sensitive binding, whereas the latter is not curvature-dependent. However, the relative contributions of both components determine the sensing of AHs. In contrast, charge density in the lipid membrane seems important primarily in attracting AHs to the membrane but does not significantly influence sensing. These observations were made possible by the ability of our assay to distinguish within our samples liposomes with and without bound protein as well as the density of bound protein. Our findings suggest that the description of membrane curvature-sensing requires consideration of several factors such as short and long range electrostatic interactions, hydrogen bonding, and the volume and structure of inserted hydrophobic residues. PMID:21953452

Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases

PubMed Central

Jadwin, Joshua A; Oh, Dongmyung; Curran, Timothy G; Ogiue-Ikeda, Mari; Jia, Lin; White, Forest M; Machida, Kazuya; Yu, Ji; Mayer, Bruce J

2016-01-01

While the affinities and specificities of SH2 domain-phosphotyrosine interactions have been well characterized, spatio-temporal changes in phosphosite availability in response to signals, and their impact on recruitment of SH2-containing proteins in vivo, are not well understood. To address this issue, we used three complementary experimental approaches to monitor phosphorylation and SH2 binding in human A431 cells stimulated with epidermal growth factor (EGF): 1) phospho-specific mass spectrometry; 2) far-Western blotting; and 3) live cell single-molecule imaging of SH2 membrane recruitment. Far-Western and MS analyses identified both well-established and previously undocumented EGF-dependent tyrosine phosphorylation and binding events, as well as dynamic changes in binding patterns over time. In comparing SH2 binding site phosphorylation with SH2 domain membrane recruitment in living cells, we found in vivo binding to be much slower. Delayed SH2 domain recruitment correlated with clustering of SH2 domain binding sites on the membrane, consistent with membrane retention via SH2 rebinding. DOI: http://dx.doi.org/10.7554/eLife.11835.001 PMID:27071344

The Polar and Electrical Nature of Dye Binding Sites on Human Red Blood Cell Membranes.

DTIC Science & Technology

positive charges at the binding sites. By increasing the concentration of the anionic BPB (or by the addition of the anionic detergent sodium lauryl ... sulfate ) these positive charges appear to be successively titrated, rendering the membrane binding sites electrically neutral at this pH. The average

Ubiquitin Interacts with the Tollip C2 and CUE Domains and Inhibits Binding of Tollip to Phosphoinositides*

PubMed Central

Mitra, Sharmistha; Traughber, C. Alicia; Brannon, Mary K.; Gomez, Stephanie; Capelluto, Daniel G. S.

2013-01-01

A large number of cellular signaling processes are directed through internalization, via endocytosis, of polyubiquitinated cargo proteins. Tollip is an adaptor protein that facilitates endosomal cargo sorting for lysosomal degradation. Tollip preferentially binds phosphatidylinositol 3-phosphate (PtdIns(3)P) via its C2 domain, an association that may be required for endosomal membrane targeting. Here, we show that Tollip binds ubiquitin through its C2 and CUE domains and that its association with the C2 domain inhibits PtdIns(3)P binding. NMR analysis demonstrates that the C2 and CUE domains bind to overlapping sites on ubiquitin, suggesting that two ubiquitin molecules associate with Tollip simultaneously. Hydrodynamic studies reveal that ubiquitin forms heterodimers with the CUE domain, indicating that the association disrupts the dimeric state of the CUE domain. We propose that, in the absence of polyubiquitinated cargo, the dual binding of ubiquitin partitions Tollip into membrane-bound and membrane-free states, a function that contributes to the engagement of Tollip in both membrane trafficking and cytosolic pathways. PMID:23880770

Supported lipid bilayer/carbon nanotube hybrids

NASA Astrophysics Data System (ADS)

Zhou, Xinjian; Moran-Mirabal, Jose M.; Craighead, Harold G.; McEuen, Paul L.

2007-03-01

Carbon nanotube transistors combine molecular-scale dimensions with excellent electronic properties, offering unique opportunities for chemical and biological sensing. Here, we form supported lipid bilayers over single-walled carbon nanotube transistors. We first study the physical properties of the nanotube/supported lipid bilayer structure using fluorescence techniques. Whereas lipid molecules can diffuse freely across the nanotube, a membrane-bound protein (tetanus toxin) sees the nanotube as a barrier. Moreover, the size of the barrier depends on the diameter of the nanotube-with larger nanotubes presenting bigger obstacles to diffusion. We then demonstrate detection of protein binding (streptavidin) to the supported lipid bilayer using the nanotube transistor as a charge sensor. This system can be used as a platform to examine the interactions of single molecules with carbon nanotubes and has many potential applications for the study of molecular recognition and other biological processes occurring at cell membranes.

Structural and Genetic Analyses of the Mycobacterium tuberculosis Protein Kinase B Sensor Domain Identify a Potential Ligand-binding Site.

PubMed

Prigozhin, Daniil M; Papavinasasundaram, Kadamba G; Baer, Christina E; Murphy, Kenan C; Moskaleva, Alisa; Chen, Tony Y; Alber, Tom; Sassetti, Christopher M

2016-10-28

Monitoring the environment with serine/threonine protein kinases is critical for growth and survival of Mycobacterium tuberculosis, a devastating human pathogen. Protein kinase B (PknB) is a transmembrane serine/threonine protein kinase that acts as an essential regulator of mycobacterial growth and division. The PknB extracellular domain (ECD) consists of four repeats homologous to penicillin-binding protein and serine/threonine kinase associated (PASTA) domains, and binds fragments of peptidoglycan. These properties suggest that PknB activity is modulated by ECD binding to peptidoglycan substructures, however, the molecular mechanisms underpinning PknB regulation remain unclear. In this study, we report structural and genetic characterization of the PknB ECD. We determined the crystal structures of overlapping ECD fragments at near atomic resolution, built a model of the full ECD, and discovered a region on the C-terminal PASTA domain that has the properties of a ligand-binding site. Hydrophobic interaction between this surface and a bound molecule of citrate was observed in a crystal structure. Our genetic analyses in M. tuberculosis showed that nonfunctional alleles were produced either by deletion of any of single PASTA domain or by mutation of individual conserved residues lining the putative ligand-binding surface of the C-terminal PASTA repeat. These results define two distinct structural features necessary for PknB signal transduction, a fully extended ECD and a conserved, membrane-distal putative ligand-binding site. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

A 23Na Multiple-Quantum-Filtered NMR Study of the Effect of the Cytoskeleton Conformation on the Anisotropic Motion of Sodium Ions in Red Blood Cells

NASA Astrophysics Data System (ADS)

Knubovets, Tatyana; Shinar, Hadassah; Eliav, Uzi; Navon, Gil

1996-01-01

Recently, it has been shown that23Na double-quantum-filtered NMR spectroscopy can be used to detect anisotropic motion of bound sodium ions in biological systems. The technique is based on the formation of the second-rank tensor when the quadrupolar interaction is not averaged to zero. Using this method, anisotropic motion of bound sodium in human and dog red blood cells was detected, and the effect was shown to depend on the integrity of the membrane cytoskeleton. In the present study, multiple-quantum-filtered techniques were applied in combination with a quadrupolar echo to measure the transverse-relaxation times,T2fandT2s. Line fitting was performed to obtain the values of the residual quadrupolar interaction, which was measured for sodium in a variety of mammalian erythrocytes of different size, shape, rheological properties, and sodium concentrations. Human unsealed white ghosts were used to study sodium bound at the anisotropic sites on the inner side of the RBC membrane. Modulations of the conformation of the cytoskeleton by the variation of either the ionic strength or pH of the suspending medium caused drastic changes in both the residual quadrupolar interaction andT2fdue to changes in the fraction of bound sodium ions as well as changes in the structure of the binding sites. By combining the two spectroscopic parameters, structural change can be followed. The changes in the structure of the sodium anisotropic binding sites deduced by this method were found to correlate with known conformational changes of the membrane cytoskeleton. Variations of the medium pH affected both the fraction of bound sodium ions and the structure of the anisotropic binding sites. Sodium and potassium were shown to bind to the anisotropic binding sites with the same affinity.

The complex nature of calcium cation interactions with phospholipid bilayers

PubMed Central

Melcrová, Adéla; Pokorna, Sarka; Pullanchery, Saranya; Kohagen, Miriam; Jurkiewicz, Piotr; Hof, Martin; Jungwirth, Pavel; Cremer, Paul S.; Cwiklik, Lukasz

2016-01-01

Understanding interactions of calcium with lipid membranes at the molecular level is of great importance in light of their involvement in calcium signaling, association of proteins with cellular membranes, and membrane fusion. We quantify these interactions in detail by employing a combination of spectroscopic methods with atomistic molecular dynamics simulations. Namely, time-resolved fluorescent spectroscopy of lipid vesicles and vibrational sum frequency spectroscopy of lipid monolayers are used to characterize local binding sites of calcium in zwitterionic and anionic model lipid assemblies, while dynamic light scattering and zeta potential measurements are employed for macroscopic characterization of lipid vesicles in calcium-containing environments. To gain additional atomic-level information, the experiments are complemented by molecular simulations that utilize an accurate force field for calcium ions with scaled charges effectively accounting for electronic polarization effects. We demonstrate that lipid membranes have substantial calcium-binding capacity, with several types of binding sites present. Significantly, the binding mode depends on calcium concentration with important implications for calcium buffering, synaptic plasticity, and protein-membrane association. PMID:27905555

The complex nature of calcium cation interactions with phospholipid bilayers

NASA Astrophysics Data System (ADS)

Melcrová, Adéla; Pokorna, Sarka; Pullanchery, Saranya; Kohagen, Miriam; Jurkiewicz, Piotr; Hof, Martin; Jungwirth, Pavel; Cremer, Paul S.; Cwiklik, Lukasz

2016-12-01

Understanding interactions of calcium with lipid membranes at the molecular level is of great importance in light of their involvement in calcium signaling, association of proteins with cellular membranes, and membrane fusion. We quantify these interactions in detail by employing a combination of spectroscopic methods with atomistic molecular dynamics simulations. Namely, time-resolved fluorescent spectroscopy of lipid vesicles and vibrational sum frequency spectroscopy of lipid monolayers are used to characterize local binding sites of calcium in zwitterionic and anionic model lipid assemblies, while dynamic light scattering and zeta potential measurements are employed for macroscopic characterization of lipid vesicles in calcium-containing environments. To gain additional atomic-level information, the experiments are complemented by molecular simulations that utilize an accurate force field for calcium ions with scaled charges effectively accounting for electronic polarization effects. We demonstrate that lipid membranes have substantial calcium-binding capacity, with several types of binding sites present. Significantly, the binding mode depends on calcium concentration with important implications for calcium buffering, synaptic plasticity, and protein-membrane association.

Continuum Approaches to Understanding Ion and Peptide Interactions with the Membrane

PubMed Central

Latorraca, Naomi R.; Callenberg, Keith M.; Boyle, Jon P.; Grabe, Michael

2014-01-01

Experimental and computational studies have shown that cellular membranes deform to stabilize the inclusion of transmembrane (TM) proteins harboring charge. Recent analysis suggests that membrane bending helps to expose charged and polar residues to the aqueous environment and polar head groups. We previously used elasticity theory to identify membrane distortions that minimize the insertion of charged TM peptides into the membrane. Here, we extend our work by showing that it also provides a novel, computationally efficient method for exploring the energetics of ion and small peptide penetration into membranes. First, we show that the continuum method accurately reproduces energy profiles and membrane shapes generated from molecular simulations of bare ion permeation at a fraction of the computational cost. Next, we demonstrate that the dependence of the ion insertion energy on the membrane thickness arises primarily from the elastic properties of the membrane. Moreover, the continuum model readily provides a free energy decomposition into components not easily determined from molecular dynamics. Finally, we show that the energetics of membrane deformation strongly depend on membrane patch size both for ions and peptides. This dependence is particularly strong for peptides based on simulations of a known amphipathic, membrane binding peptide from the human pathogen Toxoplasma gondii. In total, we address shortcomings and advantages that arise from using a variety of computational methods in distinct biological contexts. PMID:24652510

Binding of (/sup 3/H)forskolin to platelet membranes and solubilized proteins from bovine brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nelson, C.A.; Seamon, K.B.

1986-05-01

(/sup 3/H)Forskolin ((/sup 3/H)FSK) bound to platelet membranes with a Kd of 20 nM and a Bmax of 125 fmol/mg protein. The Bmax was increased to 400 fmol/mg protein in the presence of GppNHp (or NaF) and MgCl/sub 2/ with no change in Kd. PGE/sub 1/ decreased the EC50 of GppNHp to increase the Bmax for (/sup 3/H)FSK binding from 600 nM to 35 nM. In contrast, PGE/sub 1/ had no effect on the EC50 of NaF to increase (/sup 3/H)FSK binding. (/sup 3/H)FSK binding increased slowly over 60 min when forskolin and GppNHp were added to membranes simultaneously atmore » 20/sup 0/C. Preincubation of membranes with GppNHp at 20/sup 5/C also caused a linear increase in adenylate cyclase specific activity over 60 minutes. (/sup 3/H)FSK bound to solubilized protein from bovine brain membrane with a Kd of 22 nM. GppNHp increased the number of binding sites in solubilized proteins only if membranes were not preincubated with GppNHp prior to solubilization. In conclusion the number of binding sites for (/sup 3/H)FSK is increased by agents that activate adenylate cyclase through the Ns protein. These sites appear to be associated with an activated complex of the Ns protein and adenylate cyclase.« less

[Effect of amphipathic helix characteristics of FtsZ (236-245) domain on FtsZ assembly and its function in Escherichia coli strains].

PubMed

Ma, Qingsu; Zhang, Huijuan; Zheng, Xiaowei; Huo, Yujia; Lu, Feng

2017-04-04

To study the effect of amphipathic helix characteristics of FtsZ (236-245) domain on FtsZ assembly and interaction of FtsZ with FtsA in Escherichia coli strains. We constructed FtsZ and its mutant's plasmids by molecular clone and site-directed mutagenesis, and purified targeted proteins using affinity chromatography. QN23-QN29 strains were constructed by linear DNA homologous recombination and P1 transduction. We observed cellular localization patterns of FtsZ and its mutants in E. coli by living cell imaging experiments, examined membrane binding properties of FtsZ mutants by membrane proteins isolation and Western blot analysis, and analyzed interaction of FtsZ/FtsZ* with FtsA by Co-immunoprecipitation and far Western blot. Native gel separation and in vitro polymerization experiments were done to check effects of FtsZ point mutation on FtsZ assembly. Yfp-labeled FtsZE237A/K and FtsZE241A/K mutant proteins failed to localize in E. coli strains, assemble into functional Z-ring structure, and had decreased function of FtsZ (wt). In vitro experiments showed that E237A/K and E241A/K mutations of FtsZ decreased the polymerization efficiency of FtsZ monomer, weakened FtsZ*-FtsA interaction and changed membrane binding properties of FtsZ. FtsZ E237 and E241 are critical amino acids that affect the amphipathic helix characteristics of FtsZ (236-245) domain, FtsZ assembly and FtsZ-FtsA interaction in E. coli strains.

Amine Enrichment of Thin-Film Composite Membranes via Low Pressure Plasma Polymerization for Antimicrobial Adhesion.

PubMed

Reis, Rackel; Dumée, Ludovic F; He, Li; She, Fenghua; Orbell, John D; Winther-Jensen, Bjorn; Duke, Mikel C

2015-07-15

Thin-film composite membranes, primarily based on poly(amide) (PA) semipermeable materials, are nowadays the dominant technology used in pressure driven water desalination systems. Despite offering superior water permeation and salt selectivity, their surface properties, such as their charge and roughness, cannot be extensively tuned due to the intrinsic fabrication process of the membranes by interfacial polymerization. The alteration of these properties would lead to a better control of the materials surface zeta potential, which is critical to finely tune selectivity and enhance the membrane materials stability when exposed to complex industrial waste streams. Low pressure plasma was employed to introduce amine functionalities onto the PA surface of commercially available thin-film composite (TFC) membranes. Morphological changes after plasma polymerization were analyzed by SEM and AFM, and average surface roughness decreased by 29%. Amine enrichment provided isoelectric point changes from pH 3.7 to 5.2 for 5 to 15 min of plasma polymerization time. Synchrotron FTIR mappings of the amine-modified surface indicated the addition of a discrete 60 nm film to the PA layer. Furthermore, metal affinity was confirmed by the enhanced binding of silver to the modified surface, supported by an increased antimicrobial functionality with demonstrable elimination of E. coli growth. Essential salt rejection was shown minimally compromised for faster polymerization processes. Plasma polymerization is therefore a viable route to producing functional amine enriched thin-film composite PA membrane surfaces.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Band, L.; Xu, Heng; Bykov, V.

The present study demonstrates that pretreatment of rat brain membranes with (+)-cis-3-methylfentanyl ((+)-cis-MF), followed by extensive washing of the membranes, produces a wash-resistant decreasing in the binding of ({sup 3}H)-(D-ala{sup 2}, D-leu{sup 5})enkephalin to the d binding site of the opioid receptor complex ({delta}{sub cx} binding site). Intravenous administration of (+)-cis-MF (50 {mu}g/kg) to rats produced a pronounced catalepsy and also produced a wash-resistant masking of {delta}{sub cx} and {mu} binding sites in membranes prepared 120 min post-injection. Administration of 1 mg/kg i.v. of the opioid antagonist, 6-desoxy-6{beta}-fluoronaltrexone (cycloFOXY), 100 min after the injection of (+)-cis-MF (20 min prior tomore » the preparation of membranes) completely reversed the catatonia and restored masked {delta}{sub cx} binding sites to control levels. This was not observed with (+)-cycloFOXY. The implications of these and other findings for the mechanism of action of (+)-cis-MF and models of the opioid receptors are discussed.« less

Rearrangements under confinement lead to increased binding energy of Synaptotagmin-1 with anionic membranes in Mg2+ and Ca2.

PubMed

Gruget, Clémence; Coleman, Jeff; Bello, Oscar; Krishnakumar, Shyam S; Perez, Eric; Rothman, James E; Pincet, Frederic; Donaldson, Stephen H

2018-05-01

Synaptotagmin-1 (Syt1) is the primary calcium sensor (Ca 2+ ) that mediates neurotransmitter release at the synapse. The tandem C2 domains (C2A and C2B) of Syt1 exhibit functionally critical, Ca 2+ -dependent interactions with the plasma membrane. With the surface forces apparatus, we directly measure the binding energy of membrane-anchored Syt1 to an anionic membrane and find that Syt1 binds with ~6 k B T in EGTA, ~10 k B T in Mg 2+ and ~18 k B T in Ca 2+ . Molecular rearrangements measured during confinement are more prevalent in Ca 2+ and Mg 2+ and suggest that Syt1 initially binds through C2B, then reorients the C2 domains into the preferred binding configuration. These results provide energetic and mechanistic details of the Syt1 Ca 2+ -activation process in synaptic transmission. © 2018 Federation of European Biochemical Societies.

Interaction studies reveal specific recognition of an anti-inflammatory polyphosphorhydrazone dendrimer by human monocytes.

PubMed

Ledall, Jérémy; Fruchon, Séverine; Garzoni, Matteo; Pavan, Giovanni M; Caminade, Anne-Marie; Turrin, Cédric-Olivier; Blanzat, Muriel; Poupot, Rémy

2015-11-14

Dendrimers are nano-materials with perfectly defined structure and size, and multivalency properties that confer substantial advantages for biomedical applications. Previous work has shown that phosphorus-based polyphosphorhydrazone (PPH) dendrimers capped with azabisphosphonate (ABP) end groups have immuno-modulatory and anti-inflammatory properties leading to efficient therapeutic control of inflammatory diseases in animal models. These properties are mainly prompted through activation of monocytes. Here, we disclose new insights into the molecular mechanisms underlying the anti-inflammatory activation of human monocytes by ABP-capped PPH dendrimers. Following an interdisciplinary approach, we have characterized the physicochemical and biological behavior of the lead ABP dendrimer with model and cell membranes, and compared this experimental set of data to predictive computational modelling studies. The behavior of the ABP dendrimer was compared to the one of an isosteric analog dendrimer capped with twelve azabiscarboxylate (ABC) end groups instead of twelve ABP end groups. The ABC dendrimer displayed no biological activity on human monocytes, therefore it was considered as a negative control. In detail, we show that the ABP dendrimer can bind both non-specifically and specifically to the membrane of human monocytes. The specific binding leads to the internalization of the ABP dendrimer by human monocytes. On the contrary, the ABC dendrimer only interacts non-specifically with human monocytes and is not internalized. These data indicate that the bioactive ABP dendrimer is recognized by specific receptor(s) at the surface of human monocytes.

Food proteins and maturation of small intestinal microvillus membranes (MVM). I. Binding characteristics of cow's milk proteins and concanavalin A to MVM from newborn and adult rats.

PubMed

Stern, M; Gellermann, B

1988-01-01

To study maturational changes of food protein and lectin binding to rat small intestinal microvillus membranes (MVM), MVM were prepared from newborn and adult animals by a modified CaCl2 precipitation technique. Radiolabeled cow's milk proteins [alpha-lactalbumin, alpha-casein, beta-lactoglobulin, bovine serum albumin (BSA)] and the lectin concanavalin A (Con A) were used for incubations. Binding assays were done using miniature ultracentrifugation for separation of unbound material. Binding of Con A to MVM from newborn and adult rats was strong, specific, and saturable. Binding of Con A was inhibited by cold Con A and by the sugar ligand polymer mannan. Adult MVM bound more Con A than newborn preparations. Unlike Con A, binding of cow's milk proteins by MVM was weak, nonspecific, and noninhibitable. Newborn MVM bound more cow's milk proteins than adult controls. This was true for all the proteins tested (p less than 0.001). Binding rose with decreased molecular weight of cow's milk proteins, but molecular weight was not the only determining factor for binding. Trypsin treatment of MVM caused a marked increase of BSA binding in adult but not in newborn preparations. This finding indicated the importance of protein components of MVM for cow's milk protein binding. Maturational changes in protein-lipid interactions and membrane fluidity possibly influence nonspecific cow's milk protein binding to MVM. Differences in binding between newborns and adults were not directly related to maturational shifts in membrane glycosylation that are indicated by differential Con A binding. Increased cow's milk protein binding in newborn individuals might increase the potential risk to develop an adverse reaction to food proteins.

Interaction between phloretin and the red blood cell membrane

PubMed Central

1976-01-01

Phloretin binding to red blood cell components has been characterized at pH6, where binding and inhibitory potency are maximal. Binding to intact red cells and to purified hemoglobin are nonsaturated processes approximately equal in magnitude, which strongly suggests that most of the red cell binding may be ascribed to hemoglobin. This conclusion is supported by the fact that homoglobin-free red cell ghosts can bind only 10% as much phloretin as an equivalent number of red cells. The permeability of the red cell membrane to phloretin has been determined by a direct measurement at the time-course of the phloretin uptake. At a 2% hematocrit, the half time for phloretin uptake is 8.7s, corresponding to a permeability coefficient of 2 x 10(-4) cm/s. The concentration dependence of the binding to ghosts reveals two saturable components. Phloretin binds with high affinity (K diss = 1.5 muM) to about 2.5 x 10(6) sites per cell; it also binds with lower affinity (Kdiss = 54 muM) to a second (5.5 x 10(7) per cell) set of sites. In sonicated total lipid extracts of red cell ghosts, phloretin binding consists of a single, saturable component. Its affinity and total number of sites are not significantly different from those of the low affinity binding process in ghosts. No high affinity binding of phloretin is exhibited by the red cell lipid extracts. Therefore, the high affinity phloretin binding sites are related to membrane proteins, and the low affinity sites result from phloretin binding to lipid. The identification of these two types of binding sites allows phloretin effects on protein-mediated transport processes to be distinguished from effects on the lipid region of the membrane. PMID:5575

Effects of retinoids on differentiation, lipid metabolism, epidermal growth factor, and low-density lipoprotein binding in squamous carcinoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ponec, M.; Weerheim, A.; Havekes, L.

The relationship among keratinocyte differentiation capacity, lipid synthesis, low-density lipoprotein (LDL) metabolism, plasma membrane composition, and epidermal growth factor (EGF) binding has been studied in SCC-12F2 cells. The differentiation capacity of the cells, i.e., ionophore-induced cornified envelope formation, was inhibited by various retinoids and stimulated by hydrocortisone. Retinoids that caused a significant reduction of cornified envelope formation, i.e., retinoic acid and 13-cis-retinoic acid, caused only minor changes in lipid synthesis and plasma membrane composition. Arotinoid ethylsulfone, having a minor effect on cornified envelope formation, caused a drastic inhibition of cholesterol synthesis resulting in changes in the plasma membrane composition. Hydrocortisonemore » stimulated cornified envelope formation but had only minor effects on lipid synthesis and plasma membrane composition. Of all retinoids tested, only arotinoid ethylsulfone caused a drastic increase in EGF binding, while hydrocortisone had no effect. These results clearly demonstrate that the plasma membrane composition is not related to keratinocyte differentiation capacity, but most likely does determine EGF binding. Furthermore, EGF binding does not determine keratinocyte differentiation capacity.« less

Structural and mechanistic insights into phospholipid transfer by Ups1-Mdm35 in mitochondria

NASA Astrophysics Data System (ADS)

Watanabe, Yasunori; Tamura, Yasushi; Kawano, Shin; Endo, Toshiya

2015-08-01

Eukaryotic cells are compartmentalized into membrane-bounded organelles whose functions rely on lipid trafficking to achieve membrane-specific compositions of lipids. Here we focused on the Ups1-Mdm35 system, which mediates phosphatidic acid (PA) transfer between the outer and inner mitochondrial membranes, and determined the X-ray structures of Mdm35 and Ups1-Mdm35 with and without PA. The Ups1-Mdm35 complex constitutes a single domain that has a deep pocket and flexible Ω-loop lid. Structure-based mutational analyses revealed that a basic residue at the pocket bottom and the Ω-loop lid are important for PA extraction from the membrane following Ups1 binding. Ups1 binding to the membrane is enhanced by the dissociation of Mdm35. We also show that basic residues around the pocket entrance are important for Ups1 binding to the membrane and PA extraction. These results provide a structural basis for understanding the mechanism of PA transfer between mitochondrial membranes.

The RNA-Binding Site of Poliovirus 3C Protein Doubles as a Phosphoinositide-Binding Domain.

PubMed

Shengjuler, Djoshkun; Chan, Yan Mei; Sun, Simou; Moustafa, Ibrahim M; Li, Zhen-Lu; Gohara, David W; Buck, Matthias; Cremer, Paul S; Boehr, David D; Cameron, Craig E

2017-12-05

Some viruses use phosphatidylinositol phosphate (PIP) to mark membranes used for genome replication or virion assembly. PIP-binding motifs of cellular proteins do not exist in viral proteins. Molecular-docking simulations revealed a putative site of PIP binding to poliovirus (PV) 3C protein that was validated using nuclear magnetic resonance spectroscopy. The PIP-binding site was located on a highly dynamic α helix, which also functions in RNA binding. Broad PIP-binding activity was observed in solution using a fluorescence polarization assay or in the context of a lipid bilayer using an on-chip, fluorescence assay. All-atom molecular dynamics simulations of the 3C protein-membrane interface revealed PIP clustering and perhaps PIP-dependent conformations. PIP clustering was mediated by interaction with residues that interact with the RNA phosphodiester backbone. We conclude that 3C binding to membranes will be determined by PIP abundance. We suggest that the duality of function observed for 3C may extend to RNA-binding proteins of other viruses. Copyright © 2017 Elsevier Ltd. All rights reserved.

The mechanism of formation, structure and physiological relevance of covalent hemoglobin attachment to the erythrocyte membrane.

PubMed

Welbourn, Elizabeth M; Wilson, Michael T; Yusof, Ashril; Metodiev, Metodi V; Cooper, Chris E

2017-02-01

Covalent hemoglobin binding to membranes leads to band 3 (AE1) clustering and the removal of erythrocytes from the circulation; it is also implicated in blood storage lesions. Damaged hemoglobin, with the heme being in a redox and oxygen-binding inactive hemichrome form, has been implicated as the binding species. However, previous studies used strong non-physiological oxidants. In vivo hemoglobin is constantly being oxidised to methemoglobin (ferric), with around 1% of hemoglobin being in this form at any one time. In this study we tested the ability of the natural oxidised form of hemoglobin (methemoglobin) in the presence or absence of the physiological oxidant hydrogen peroxide to initiate membrane binding. The higher the oxidation state of hemoglobin (from Fe(III) to Fe(V)) the more binding was observed, with approximately 50% of this binding requiring reactive sulphydryl groups. The hemoglobin bound was in a high molecular weight complex containing spectrin, ankyrin and band 4.2, which are common to one of the cytoskeletal nodes. Unusually, we showed that hemoglobin bound in this way was redox active and capable of ligand binding. It can initiate lipid peroxidation showing the potential to cause cell damage. In vivo oxidative stress studies using extreme endurance exercise challenges showed an increase in hemoglobin membrane binding, especially in older cells with lower levels of antioxidant enzymes. These are then targeted for destruction. We propose a model where mild oxidative stress initiates the binding of redox active hemoglobin to the membrane. The maximum lifetime of the erythrocyte is thus governed by the redox activity of the cell; from the moment of its release into the circulation the timer is set. Copyright © 2016. Published by Elsevier Inc.

The interaction of substituted benzamides with brain benzodiazepine binding sites in vitro.

PubMed Central

Horton, R. W.; Lowther, S.; Chivers, J.; Jenner, P.; Marsden, C. D.; Testa, B.

1988-01-01

1. The interaction of substituted benzamides with brain benzodiazepine (BDZ) binding sites was examined by their ability to displace [3H]-flunitrazepam ([3H]-FNM) from specific binding sites in bovine cortical membranes in vitro. 2. Clebopride, Delagrange 2674, Delagrange 2335 and BRL 20627 displayed concentration-dependent displacement of [3H]-FNM with IC50 values of 73 nM, 132 nM, 7.7 microM and 5.9 microM, respectively. Other substituted benzamides including metoclopramide, sulpiride, tiapride, sultopride and cisapride were inactive at 10(-5) M. 3. Inhibition by clebopride and Delagrange 2674 of [3H]-FNM binding was apparently competitive and readily reversible. 4. In the presence of gamma-aminobutyric acid (GABA), the ability of diazepam and Delagrange 2674 to displace [3H]-Ro 15-1788 binding was increased 3.6 and 1.6 fold respectively, compared to the absence of GABA, while ethyl beta-carboline-3-carboxylate (beta CCE) and clebopride were less potent in the presence of GABA. 5. Diazepam was 30 fold less potent at displacing [3H]-Ro 15-1788 in membranes that had been photoaffinity labelled with FNM than in control membranes, whereas the potency of beta CCE did not differ. Clebopride and Delagrange 2674 showed a less than two fold loss of potency in photoaffinity labelled membranes. 6. The pattern of binding of clebopride and Delagrange 2674 in these in vitro tests is similar to that found previously with partial agonists or antagonists at BDZ binding sites. 7. Clebopride and Delagrange 2674 inhibited [3H]-FNM binding with similar potency in rat cerebellar and hippocampal membranes, suggesting they have no selectivity for BDZ1 and BDZ2 binding sites.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2850059

Studies on the uptake of fatty acids by brush border membranes of the rabbit intestine.

PubMed

Proulx, P; Aubry, H; Brglez, I; Williamson, D G

1985-04-01

Initial studies revealed that the uptake of palmitic acid and oleic acid into brush border membranes was similar when these were isolated from either whole small intestine, jejunum, or ileum. The uptake of these fatty acids was somewhat lower with membranes obtained from duodenum. Subsequent studies, all with membranes obtained from whole intestine, indicated an increase in binding with chain length of fatty acid of up to 16 carbons. Unsaturation decreased this uptake somewhat. Taurocholate and 1-palmitoyl lysolecithin had a moderate stimulatory effect on the binding of oleic acid and palmitic acid at concentrations of 10 and 0.5 mM, respectively, and inhibited at higher concentrations. Addition of 1.4 mM egg lecithin to the fatty acid - bile salt micelles, such that the lecithin - bile salt ratio was 0.2, decreased the uptake of fatty acids generally, but did not significantly affect the pattern of binding by membrane fractions isolated from different segments nor did it change the pattern of labelling when fatty acid chain length and unsaturation were varied. At lower concentrations, egg lecithin had little effect on the uptake of oleic acid, whereas dipalmitoyl phosphatidylcholine stimulated binding of both palmitic acid and oleic acid over the entire range of concentrations tested. Preincubation of the membranes with this saturated phospholipid stimulated the uptake of oleic acid, and addition of this choline lipid to the oleic acid - bile salt containing micelles did not substantially enhance fatty acid uptake in lipid-treated membranes. The binding of fatty acid was very rapid either in the presence or the absence of Ca2+, such that even in zero-time controls essentially equilibrium bindings were obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma membrane order and fluidity are diversely triggered by elicitors of plant defence.

PubMed

Sandor, Roman; Der, Christophe; Grosjean, Kevin; Anca, Iulia; Noirot, Elodie; Leborgne-Castel, Nathalie; Lochman, Jan; Simon-Plas, Françoise; Gerbeau-Pissot, Patricia

2016-09-01

Although plants are exposed to a great number of pathogens, they usually defend themselves by triggering mechanisms able to limit disease development. Alongside signalling events common to most such incompatible interactions, modifications of plasma membrane (PM) physical properties could be new players in the cell transduction cascade. Different pairs of elicitors (cryptogein, oligogalacturonides, and flagellin) and plant cells (tobacco and Arabidopsis) were used to address the issue of possible modifications of plant PM biophysical properties induced by elicitors and their links to other events of the defence signalling cascade. We observed an increase of PM order whatever the elicitor/plant cell pair used, provided that a signalling cascade was induced. Such membrane modification is dependent on the NADPH oxidase-mediated reactive oxygen species production. Moreover, cryptogein, which is the sole elicitor able to trap sterols, is also the only one able to trigger an increase in PM fluidity. The use of cryptogein variants with altered sterol-binding properties confirms the strong correlation between sterol removal from the PM and PM fluidity enhancement. These results propose PM dynamics as a player in early signalling processes triggered by elicitors of plant defence. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

The phosphatidylinositol transfer protein RdgBβ binds 14-3-3 via its unstructured C-terminus, whereas its lipid-binding domain interacts with the integral membrane protein ATRAP (angiotensin II type I receptor-associated protein).

PubMed

Garner, Kathryn; Li, Michelle; Ugwuanya, Natalie; Cockcroft, Shamshad

2011-10-01

PITPs [PI (phosphatidylinositol) transfer proteins] bind and transfer PI between intracellular membranes and participate in many cellular processes including signalling, lipid metabolism and membrane traffic. The largely uncharacterized PITP RdgBβ (PITPNC1; retinal degeneration type B β), contains a long C-terminal disordered region following its defining N-terminal PITP domain. In the present study we report that the C-terminus contains two tandem phosphorylated binding sites (Ser(274) and Ser(299)) for 14-3-3. The C-terminus also contains PEST sequences which are shielded by 14-3-3 binding. Like many proteins containing PEST sequences, the levels of RdgBβ are regulated by proteolysis. RdgBβ is degraded with a half-life of 4 h following ubiquitination via the proteasome. A mutant RdgBβ which is unable to bind 14-3-3 is degraded even faster with a half-life of 2 h. In vitro, RdgBβ is 100-fold less active than PITPα for PI transfer, and RdgBβ proteins (wild-type and a mutant that cannot bind 14-3-3) expressed in COS-7 cells or endogenous proteins from heart cytosol do not exhibit transfer activity. When cells are treated with PMA, the PITP domain of RdgBβ interacts with the integral membrane protein ATRAP (angiotensin II type I receptor-associated protein; also known as AGTRAP) causing membrane recruitment. We suggest that RdgBβ executes its function following recruitment to membranes via its PITP domain and the C-terminal end of the protein could regulate entry to the hydrophobic cavity.

Exceptionally tight membrane-binding may explain the key role of the synaptotagmin-7 C 2 A domain in asynchronous neurotransmitter release

DOE Office of Scientific and Technical Information (OSTI.GOV)

Voleti, Rashmi; Tomchick, Diana R.; Südhof, Thomas C.

Synaptotagmins (Syts) act as Ca2+ sensors in neurotransmitter release by virtue of Ca2+-binding to their two C2 domains, but their mechanisms of action remain unclear. Puzzlingly, Ca2+-binding to the C2B domain appears to dominate Syt1 function in synchronous release, whereas Ca2+-binding to the C2A domain mediates Syt7 function in asynchronous release. Here we show that crystal structures of the Syt7 C2A domain and C2AB region, and analyses of intrinsic Ca2+-binding to the Syt7 C2 domains using isothermal titration calorimetry, did not reveal major differences that could explain functional differentiation between Syt7 and Syt1. However, using liposome titrations under Ca2+ saturatingmore » conditions, we show that the Syt7 C2A domain has a very high membrane affinity and dominates phospholipid binding to Syt7 in the presence or absence of L-α-phosphatidylinositol 4,5-diphosphate (PIP2). For Syt1, the two Ca2+-saturated C2 domains have similar affinities for membranes lacking PIP2, but the C2B domain dominates binding to PIP2-containing membranes. Mutagenesis revealed that the dramatic differences in membrane affinity between the Syt1 and Syt7 C2A domains arise in part from apparently conservative residue substitutions, showing how striking biochemical and functional differences can result from the cumulative effects of subtle residue substitutions. Viewed together, our results suggest that membrane affinity may be a key determinant of the functions of Syt C2 domains in neurotransmitter release.« less

Functional interaction between the two halves of the photoreceptor-specific ATP binding cassette protein ABCR (ABCA4). Evidence for a non-exchangeable ADP in the first nucleotide binding domain.

PubMed

Ahn, Jinhi; Beharry, Seelochan; Molday, Laurie L; Molday, Robert S

2003-10-10

ABCR, also known as ABCA4, is a member of the superfamily of ATP binding cassette transporters that is believed to transport retinal or retinylidene-phosphatidylethanolamine across photoreceptor disk membranes. Mutations in the ABCR gene are responsible for Stargardt macular dystrophy and related retinal dystrophies that cause severe loss in vision. ABCR consists of two tandemly arranged halves each containing a membrane spanning segment followed by a large extracellular/lumen domain, a multi-spanning membrane domain, and a nucleotide binding domain (NBD). To define the role of each NBD, we examined the nucleotide binding and ATPase activities of the N and C halves of ABCR individually and co-expressed in COS-1 cells and derived from trypsin-cleaved ABCR in disk membranes. When disk membranes or membranes from co-transfected cells were photoaffinity labeled with 8-azido-ATP and 8-azido-ADP, only the NBD2 in the C-half bound and trapped the nucleotide. Co-expressed half-molecules displayed basal and retinal-stimulated ATPase activity similar to full-length ABCR. The individually expressed N-half displayed weak 8-azido-ATP labeling and low basal ATPase activity that was not stimulated by retinal, whereas the C-half did not bind ATP and exhibited little if any ATPase activity. Purified ABCR contained one tightly bound ADP, presumably in NBD1. Our results indicate that only NBD2 of ABCR binds and hydrolyzes ATP in the presence or absence of retinal. NBD1, containing a bound ADP, associates with NBD2 to play a crucial, non-catalytic role in ABCR function.

Passive transport and binding of lead by human red blood cells.

PubMed

Simons, T J

1986-09-01

The uptake of Pb into human red blood cells has been studied using Pb buffers. Passive Pb movements can be studied conveniently when the cells are depleted of adenosine 5'-triphosphate (ATP), to eliminate active transport, and of inorganic phosphate, to prevent precipitation of lead phosphate. Pb can cross the membrane passively in either direction. Influx and efflux show similar properties. Passive Pb transport is strongly stimulated by HCO3-, and is reduced by replacing Cl- with ClO4-. It is inhibited by low concentrations of 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS) and 4,4'-diisothiocyanostilbene-2.2'-disulphonic acid (DIDS), characteristic inhibitors of anion transport. Pb uptake is unaffected by varying the external concentrations of Na+, K+ and Ca2+. When Pb enters the cell, it binds mainly to haemoglobin. The ratio of bound Pb:free Pb2+ in the cytosol is estimated to be 6000:1. Pb binding to haemoglobin is unaffected by oxygenation. Binding to albumin is quantitatively similar to binding to haemoglobin. The implications of these results for the transport and binding of Pb in the blood are discussed.

Specific binding of 15 HETE to lymphocytes. Effects on the fluidity of plasmatic membranes.

PubMed

Mexmain, S; Gualde, N; Aldigier, J C; Motta, C; Chable-Rabinovitch, H; Rigaud, M

1984-01-01

Specific binding of mouse lymphocytes for 15 HETE was examined by incubating cells with [14C]-15 HETE, 1 X 10(-8) to 1 X 10(-10)M. It was observed that the specific binding of radiolabeled 15 HETE is a function of time, of temperature and is modified by Ca2+ and dithiothreitol. When a fluorescent probe was embedded in the phospholipid core of the lymphocyte membrane and its motion analysed by fluorescence polarization, it was observed that 15 HETE increases the viscosity of the plasmatic membrane.

GSL-enriched membrane microdomains in innate immune responses.

PubMed

Nakayama, Hitoshi; Ogawa, Hideoki; Takamori, Kenji; Iwabuchi, Kazuhisa

2013-06-01

Many pathogens target glycosphingolipids (GSLs), which, together with cholesterol, GPI-anchored proteins, and various signaling molecules, cluster on host cell membranes to form GSL-enriched membrane microdomains (lipid rafts). These GSL-enriched membrane microdomains may therefore be involved in host-pathogen interactions. Innate immune responses are triggered by the association of pathogens with phagocytes, such as neutrophils, macrophages and dendritic cells. Phagocytes express a diverse array of pattern-recognition receptors (PRRs), which sense invading microorganisms and trigger pathogen-specific signaling. PRRs can recognize highly conserved pathogen-associated molecular patterns expressed on microorganisms. The GSL lactosylceramide (LacCer, CDw17), which binds to various microorganisms, including Candida albicans, is expressed predominantly on the plasma membranes of human mature neutrophils and forms membrane microdomains together with the Src family tyrosine kinase Lyn. These LacCer-enriched membrane microdomains can mediate superoxide generation, migration, and phagocytosis, indicating that LacCer functions as a PRR in innate immunity. Moreover, the interactions of GSL-enriched membrane microdomains with membrane proteins, such as growth factor receptors, are important in mediating the physiological properties of these proteins. Similarly, we recently found that interactions between LacCer-enriched membrane microdomains and CD11b/CD18 (Mac-1, CR3, or αMβ2-integrin) are significant for neutrophil phagocytosis of non-opsonized microorganisms. This review describes the functional role of LacCer-enriched membrane microdomains and their interactions with CD11b/CD18.

[Passage of nonsteroidal anti-inflammatory agents across the synovial membrane].

PubMed

Netter, P; Bannwarth, B; Monot, C; Royer, R J; Gaucher, A

1983-09-24

The therapeutic effectiveness of non-steroid anti-inflammatory (NSAI) drugs is partly determined by their passage across the synovial membrane. The synovium can be compared to a double barrier the permeability of which to NSAI drugs depends on the degree of inflammation of the joint and on the pharmacokinetic properties of the drugs (lipophilia, pka, protein-binding). A few hours after one single systemic dose, concentrations in the synovial fluid are higher than in serum. During chronic administration, concentrations of NSAI drugs with a short half-life vary less in synovial fluid than in serum. During steady state, free fractions of NSAI drugs with prolonged half-life may be similar in both compartments.

The membrane bound bacterial lipocalin Blc is a functional dimer with binding preference for lysophospholipids

PubMed Central

Campanacci, Valérie; Bishop, Russell E.; Blangy, Stéphanie; Tegoni, Mariella; Cambillau, Christian

2016-01-01

Lipocalins, a widespread multifunctional family of small proteins (15–25 kDa) have been first described in eukaryotes and more recently in Gram-negative bacteria. Bacterial lipocalins belonging to class I are outer membrane lipoproteins, among which Blc from E. coli is the better studied. Blc is expressed under conditions of starvation and high osmolarity, conditions known to exert stress on the cell envelope. The structure of Blc that we have previously solved (V. Campanacci, D. Nurizzo, S. Spinelli, C. Valencia, M. Tegoni, C. Cambillau, FEBS Lett. 562 (2004) 183–188.) suggested its possible role in binding fatty acids or phospholipids. Both physiological and structural data on Blc, therefore, point to a role in storage or transport of lipids necessary for membrane maintenance. In order to further document this hypothesis for Blc function, we have performed binding studies using fluorescence quenching experiments. Our results indicate that dimeric Blc binds fatty acids and phospholipids in a micromolar Kd range. The crystal structure of Blc with vaccenic acid, an unsaturated C18 fatty acid, reveals that the binding site spans across the Blc dimer, opposite to its membrane anchored face. An exposed unfilled pocket seemingly suited to bind a polar group attached to the fatty acid prompted us to investigate lyso-phospholipids, which were found to bind in a nanomolar Kd range. We discuss these findings in terms of a potential role for Blc in the metabolism of lysophospholipids generated in the bacterial outer membrane. PMID:16920109

Evidence for a G protein-coupled diadenosine-5',5'''-P1,P4-tetraphosphate (Ap4A) receptor binding site in lung membranes from rat.

PubMed

Laubinger, W; Reiser, G

1999-01-29

Nucleotide receptors are of considerable importance in the treatment of lung diseases, such as cystic fibrosis. Because diadenosine polyphosphates may also be of significance as signalling molecules in lung, as they are in a variety of tissues, in the present work we investigated the binding sites for [3H]diadenosine-5',5'''-P1,P4-tetraphosphate (Ap4A) in plasma membranes from rat lung and studied their possible coupling to G proteins. We present evidence for a single high-affinity binding site for [3H]Ap4A with similar affinity for other diadenosine polyphosphates ApnA (n = 2 to 6). Displacement studies with different nucleotides revealed that the [3H]Ap4A binding site was different from P2X and P2Y2 receptor binding sites. Pretreatment of lung membranes with GTPgammaS or GTP in the presence of Mg2+ increased the Ki for Ap4A from 91 nM to 5.1 microM, which is indicative of G protein coupling. The putative coupling to G proteins was further confirmed by the enhancement of [35S]GTPgammaS binding (to Galpha proteins) to lung membranes by Ap4A (63% increase over basal) in a concentration-dependent manner. Therefore, our data for the first time provide evidence of a G protein-coupled Ap4A binding site in lung membranes.

Interplay between membrane curvature and protein conformational equilibrium investigated by solid-state NMR.

PubMed

Liao, Shu Y; Lee, Myungwoon; Hong, Mei

2018-03-01

Many membrane proteins sense and induce membrane curvature for function, but structural information about how proteins modulate their structures to cause membrane curvature is sparse. We review our recent solid-state NMR studies of two virus membrane proteins whose conformational equilibrium is tightly coupled to membrane curvature. The influenza M2 proton channel has a drug-binding site in the transmembrane (TM) pore. Previous chemical shift data indicated that this pore-binding site is lost in an M2 construct that contains the TM domain and a curvature-inducing amphipathic helix. We have now obtained chemical shift perturbation, protein-drug proximity, and drug orientation data that indicate that the pore-binding site is restored when the full cytoplasmic domain is present. This finding indicates that the curvature-inducing amphipathic helix distorts the TM structure to interfere with drug binding, while the cytoplasmic tail attenuates this effect. In the second example, we review our studies of a parainfluenza virus fusion protein that merges the cell membrane and the virus envelope during virus entry. Chemical shifts of two hydrophobic domains of the protein indicate that both domains have membrane-dependent backbone conformations, with the β-strand structure dominating in negative-curvature phosphatidylethanolamine (PE) membranes. 31 P NMR spectra and 1 H- 31 P correlation spectra indicate that the β-strand-rich conformation induces saddle-splay curvature to PE membranes and dehydrates them, thus stabilizing the hemifusion state. These results highlight the indispensable role of solid-state NMR to simultaneously determine membrane protein structures and characterize the membrane curvature in which these protein structures exist. Copyright © 2018 Elsevier Inc. All rights reserved.

Phosphatidic acid - a simple phospholipid with multiple faces.

PubMed

Zegarlińska, Jolanta; Piaścik, Magda; Sikorski, Aleksander F; Czogalla, Aleksander

2018-01-01

Phosphatidic acid (PA) is the simplest glycerophospholipid naturally occurring in living organisms, and even though its content among other cellular lipids is minor, it is drawing more and more attention due to its multiple biological functions. PA is a precursor for other phospholipids, acts as a lipid second messenger and, due to its structural properties, is also a modulator of membrane shape. Although much is known about interaction of PA with its effectors, the molecular mechanisms remain unresolved to a large degree. Throughout many of the well-characterized PA cellular sensors, no conserved binding domain can be recognized. Moreover, not much is known about the cellular dynamics of PA and how it is distributed among subcellular compartments. Remarkably, PA can play distinct roles within each of these compartments. For example, in the nucleus it behaves as a mitogen, influencing gene expression regulation, and in the Golgi membrane it plays a role in membrane trafficking. Here, we discuss how a biophysical experimental approach enabled PA behavior to be described in the context of a lipid bilayer and to what extent various physicochemical conditions may modulate the functional properties of this lipid. Understanding these aspects would help to unravel specific mechanisms of PA-driven membrane transformations and protein recruitment and thus would lead to a clearer picture of the biological role of PA.

Surface dynamics of aerolysin on the plasma membrane of living cells.

PubMed

Abrami, L; Fivaz, M; van der Goot, F G

2000-10-01

Aerolysin secreted by the human pathogen Aeromonas hydrophila belongs to a group of bacterial toxins that are hemolytic and form channels in biological membranes. The toxin is secreted as an inactive precursor proaerolysin that must be proteolytically processed at its C-terminus to become active. The toxin then polymerizes into a heptameric ring that is amphipathic and can insert into a lipid bilayer and form a pore. We have examined these various steps at the surface of target cells. The toxin binds to specific receptors. Various receptors have been identified, all of which are anchored to the plasma membrane via a glycosylphosphatidyl inositol (GPI)-anchored moiety. The GPI anchor confers to the protein that is linked to it two usual properties: (i) the protein has a higher lateral mobility in a phospholipid bilayer than its transmembrane counterpart, (ii) the protein has the capacity to transiently associate with cholesterol-glycosphingolipid-rich microdomains. We have shown that both these properties of GPI-anchored proteins are exploited by proaerolysin bound to its receptor. The high lateral mobility within the phosphoglyceride region of the plasma membrane favors the encounter of the protoxin with its converting enzyme furin. The ability to associate with microdomains on the other hand favors the oligomerization process presumably by concentrating the toxin locally.

Avian pathogenic Escherichia coli bind fibronectin and laminin.

PubMed

Ramírez, Rosa María; Almanza, Yolanda; González, Rafael; García, Santos; Heredia, Norma

2009-04-01

Avian colisepticemia frequently occurs after respiratory tract damage, the primary site for infection allows bacteria to encounter an exposed basement membrane, where laminin and fibronectin are important components. We investigated the ability of an isolate of avian pathogenic Escherichia coli to bind fibronectin and laminin. Using Far-western dot blot analysis, we demonstrated the ability of this microorganism to bind basement membrane proteins fibronectin and laminin. Results from an ELISA-based approach indicate that the binding to these membrane proteins was bacterial-dose dependent. Furthermore, two specific E. coli polypeptides, of 32 kDa and 130 kDa, reacted with laminin and fibronectin, respectively. Further evaluation of these potential bacterial adhesins may provide insights into the pathogenesis of colibacillosis.

Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily

PubMed Central

Lenoir, Marc; Kufareva, Irina; Abagyan, Ruben; Overduin, Michael

2015-01-01

The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH) domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH) and Tec homology (TH) domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA) program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer. PMID:26512702

[Glutamate-binding membrane proteins from human platelets].

PubMed

Gurevich, V S; Popov, Iu G; Gorodinskiĭ, A I; Dambinova, S A

1991-09-01

Solubilization of the total membrane fraction of human platelets in a 2% solution of sodium deoxycholate and subsequent affinity chromatography on glutamate agarose resulted in two protein fractions possessing a glutamate-binding activity. As can be evidenced from radioligand binding data, the first fraction contains two types of binding sites (Kd1 = 1 microM, Bmax 1 = 100 pmol/mg of protein; Kd2 = 9.3 microMm Bmax2 = 395 pmol/mg of protein). The second fraction has only one type of binding sites (Kd = 1 microM, Bmax = = 110 pmol/mg of protein). SDS-PAAG electrophoresis revealed the presence in the first fraction of proteins with Mr of 14, 24, 56 and 155 kDa, whereas the second fraction was found to contain 14, 46, 71 and 155 kDa proteins. Solid phase immunoenzymatic analysis using poly- and monoclonal specific antibodies against mammalian brain glutamate-binding proteins revealed a marked immunochemical similarity of the isolated protein fractions with human brain synaptic membrane glutamate-binding proteins.

Highly potent antimicrobial peptides from N-terminal membrane-binding region of E. coli MreB.

PubMed

Saikia, Karabi; Sravani, Yalavarthi Durga; Ramakrishnan, Vibin; Chaudhary, Nitin

2017-02-23

Microbial pathogenesis is a serious health concern. The threat escalates as the existing conventional antimicrobials are losing their efficacy against the evolving pathogens. Peptides hold promise to be developed into next-generation antibiotics. Antimicrobial peptides adopt amphipathic structures that could selectively bind to and disrupt the microbial membranes. Interaction of proteins with membranes is central to all living systems and we reasoned that the membrane-binding domains in microbial proteins could be developed into efficient antimicrobials. This is an interesting approach as self-like sequences could elude the microbial strategies of degrading the antimicrobial peptides, one of the mechanisms of showing resistance to antimicrobials. We selected the 9-residue-long membrane-binding region of E. coli MreB protein. The 9-residue peptide (C-terminal amide) and its N-terminal acetylated analog displayed broad-spectrum activity, killing Gram-negative bacteria, Gram-positive bacteria, and fungi. Extension with a tryptophan residue at the N-terminus drastically improved the activity of the peptides with lethal concentrations ≤10 μM against all the organisms tested. The tryptophan-extended peptides caused complete killing of C. albicans as well as gentamicin and methicillin resistant S. aureus at 5 μM concentration. Lipid-binding studies and electron microscopic analyses of the peptide-treated microbes suggest membrane disruption as the mechanism of killing.

Theoretical studies of Pt-Ti nanoparticles for potential use as PEMFC electrocatalysts.

PubMed

Jennings, Paul C; Pollet, Bruno G; Johnston, Roy L

2012-03-07

A theoretical investigation is presented of alloying platinum with titanium to form binary Pt-Ti nanoalloys as an alternative to the expensive pure platinum catalysts commonly used for Proton Exchange Membrane Fuel Cell cathode electrocatalysts. Density Functional Theory calculations are performed to investigate compositional effects on structural properties as well as Oxygen Reduction Reaction kinetics and poisoning effects. High symmetry A(32)-B(6) clusters are studied to investigate structural properties. From these structures binding energies of hydroxyl and carbon monoxide are studied on a range of sites on the surface of the clusters. Promising results are obtained suggesting that the bimetallic Pt-Ti nanoalloys may exhibit enhanced properties compared to pure platinum catalysts.

Dimensionality of Motion and Binding Valency Govern Receptor-Ligand Kinetics As Revealed by Agent-Based Modeling.

PubMed

Lehnert, Teresa; Figge, Marc Thilo

2017-01-01

Mathematical modeling and computer simulations have become an integral part of modern biological research. The strength of theoretical approaches is in the simplification of complex biological systems. We here consider the general problem of receptor-ligand binding in the context of antibody-antigen binding. On the one hand, we establish a quantitative mapping between macroscopic binding rates of a deterministic differential equation model and their microscopic equivalents as obtained from simulating the spatiotemporal binding kinetics by stochastic agent-based models. On the other hand, we investigate the impact of various properties of B cell-derived receptors-such as their dimensionality of motion, morphology, and binding valency-on the receptor-ligand binding kinetics. To this end, we implemented an algorithm that simulates antigen binding by B cell-derived receptors with a Y-shaped morphology that can move in different dimensionalities, i.e., either as membrane-anchored receptors or as soluble receptors. The mapping of the macroscopic and microscopic binding rates allowed us to quantitatively compare different agent-based model variants for the different types of B cell-derived receptors. Our results indicate that the dimensionality of motion governs the binding kinetics and that this predominant impact is quantitatively compensated by the bivalency of these receptors.

Dimensionality of Motion and Binding Valency Govern Receptor–Ligand Kinetics As Revealed by Agent-Based Modeling

PubMed Central

Lehnert, Teresa; Figge, Marc Thilo

2017-01-01

Mathematical modeling and computer simulations have become an integral part of modern biological research. The strength of theoretical approaches is in the simplification of complex biological systems. We here consider the general problem of receptor–ligand binding in the context of antibody–antigen binding. On the one hand, we establish a quantitative mapping between macroscopic binding rates of a deterministic differential equation model and their microscopic equivalents as obtained from simulating the spatiotemporal binding kinetics by stochastic agent-based models. On the other hand, we investigate the impact of various properties of B cell-derived receptors—such as their dimensionality of motion, morphology, and binding valency—on the receptor–ligand binding kinetics. To this end, we implemented an algorithm that simulates antigen binding by B cell-derived receptors with a Y-shaped morphology that can move in different dimensionalities, i.e., either as membrane-anchored receptors or as soluble receptors. The mapping of the macroscopic and microscopic binding rates allowed us to quantitatively compare different agent-based model variants for the different types of B cell-derived receptors. Our results indicate that the dimensionality of motion governs the binding kinetics and that this predominant impact is quantitatively compensated by the bivalency of these receptors. PMID:29250071

Formation and Characterization of Langmuir Silk Films

DTIC Science & Technology

1993-07-01

studied for phase transitions as immo- fibroin and sericin . Fibroin is the protein that forms the bilization matrices have been in the form of cast mem...properties. Sericins are a group membrane materials. However, the casting process has of gummy proteins which bind the fibroin filaments, limitations...approximately 5 mm X 5 mm pieces. The cocoon material was boiled in distilled water for 2 h with changes 7- of water to solubilize and remove the sericin

Design of an optically stable pH sensor based on immobilization of Giemsa on triacetylcellulose membrane.

PubMed

Khodadoust, Saeid; Kouri, Narges Cham; Talebiyanpoor, Mohammad Sharif; Deris, Jamile; Pebdani, Arezou Amiri

2015-12-01

In this work a simple, inexpensive, and sensitive optical sensor based on triacetylcellulose membrane as solid support was developed by using immobilization of Giemsa indicator for pH measurement. In this method, the influence variables on the membrane performance including pH concentration of indicator, response time, ionic strength, and reversibility were investigated. At optimum values of all variables the response of optical pH sensor is linear in the pH range of 3.0-12.0. This optical sensor was produced through simultaneous binding of the Giemsa on the activated triacetylcellulose membrane which responded to the pH changes in a broader linear range within less than 2.0 min and suitable reproducibility (RSD<5%). Stability results showed that this sensor was stable after 6 months of storage in the water/ethanol (50:50, v/v) solution without any measurable divergence in response properties (less than 5% RSD). Copyright © 2015 Elsevier B.V. All rights reserved.

Characterization of a monoclonal antibody against P57, the C3/C3b-cleaving proteinase expressed in human erythrocyte membranes.

PubMed

Fiandino-Tirel, A; Barel, M; Lyamani, F; Gauffre, A; Hermann, J; Frade, R

1991-08-01

A monoclonal antibody was raised against p57, a serine proteinase, characterized by an apparent molecular weight of 57 kDa, and purified from human erythrocyte membranes. P57 proteinase cleaves the human third component of complement, C3. The antibody selected, MP1, of IgG2a isotype, precipitated specifically the p57 antigen which carried the C3/C3b-cleaving activity present in membrane crude extract of human erythrocytes. P57 proteinase eluted from MP1-sepharose was inhibited by 5 x 10(-4) M PMSF, enhanced by 0.5% SDS and generated C3 fragments identical to those generated by membrane crude extract of human erythrocytes. All these properties were identical to those of the p57 previously purified by biochemical procedures. In addition, 5000 binding sites were detected on cell surface. This MP1 monoclonal antibody will be helpful to analyse the role of p57 in human erythrocytes.

Peptidoglycan-associated outer membrane protein Mep45 of rumen anaerobe Selenomonas ruminantium forms a non-specific diffusion pore via its C-terminal transmembrane domain.

PubMed

Kojima, Seiji; Hayashi, Kanako; Tochigi, Saeko; Kusano, Tomonobu; Kaneko, Jun; Kamio, Yoshiyuki

2016-10-01

The major outer membrane protein Mep45 of Selenomonas ruminantium, an anaerobic Gram-negative bacterium, comprises two distinct domains: the N-terminal S-layer homologous (SLH) domain that protrudes into the periplasm and binds to peptidoglycan, and the remaining C-terminal transmembrane domain, whose function has been unknown. Here, we solubilized and purified Mep45 and characterized its function using proteoliposomes reconstituted with Mep45. We found that Mep45 forms a nonspecific diffusion channel via its C-terminal region. The channel was permeable to solutes smaller than a molecular weight of roughly 600, and the estimated pore radius was 0.58 nm. Truncation of the SLH domain did not affect the channel property. On the basis of the fact that Mep45 is the most abundant outer membrane protein in S. ruminantium, we conclude that Mep45 serves as a main pathway through which small solutes diffuse across the outer membrane of this bacterium.

Design of a homogeneous multifunctional supported lipid membrane on layer-by-layer coated microcarriers.

PubMed

Göse, Martin; Pescador, Paula; Reibetanz, Uta

2015-03-09

Key challenges in the development of drug delivery systems are the prevention of serum compartment interaction and the targeted delivery of the cargo. Layer-by-Layer microcarriers offer many advantages due to various options in drug assembly and multifunctional design. Surface modification with a supported lipid membrane enhances biocompatibility, drug protection ability, and specific functionality. However, the integration of functionalized lipids strongly influences the membrane formation and is often accompanied by submicrometer irregularities: The accessibility of underlying polymers to serum components may change the carrier's properties and enhances the susceptibility to opsonization. Therefore, the formation of a tightly assembled multifunctional lipid membrane has been emphasized. A phosphatidylserine/phosphatidylcholine (POPS/POPC) bilayer equipped with phosphatidylethanolamine-polyethylene glycol-biotin (PE-PEG-Biotin) was used to facilitate a biotin/streptavidin binding site for a variable attachment of an additional function, such as antibodies for specific targeting. Thus, a prefunctionalized carrier where only the outer functionality needs to be replaced without disturbing the underlying structure could be created.

Thermodynamics of melittin binding to lipid bilayers. Aggregation and pore formation.

PubMed

Klocek, Gabriela; Schulthess, Therese; Shai, Yechiel; Seelig, Joachim

2009-03-31

Lipid membranes act as catalysts for protein folding. Both alpha-helical and beta-sheet structures can be induced by the interaction of peptides or proteins with lipid surfaces. Melittin, the main component of bee venom, is a particularly well-studied example for the membrane-induced random coil-to-alpha-helix transition. Melittin in water adopts essentially a random coil conformation. The cationic amphipathic molecule has a high affinity for neutral and anionic lipid membranes and exhibits approximately 50-65% alpha-helix conformation in the membrane-bound state. At higher melittin concentrations, the peptide forms aggregates or pores in the membrane. In spite of the long-standing interest in melittin-lipid interactions, no systematic thermodynamic study is available. This is probably caused by the complexity of the binding process. Melittin binding to lipid vesicles is fast and occurs within milliseconds, but the binding process involves at least four steps, namely, (i) the electrostatic attraction of the cationic peptide to an anionic membrane surface, (ii) the hydrophobic insertion into the lipid membrane, (iii) the conformational change from random coil to alpha-helix, and (iv) peptide aggregation in the lipid phase. We have combined microelectrophoresis (measurement of the zeta potential), isothermal titration calorimetry, and circular dichroism spectroscopy to provide a thermodynamic analysis of the individual binding steps. We have compared melittin with a synthetic analogue, [D]-V(5,8),I(17),K(21)-melittin, for which alpha-helix formation is suppressed and replaced by beta-structure formation. The comparison reveals that the thermodynamic parameters for the membrane-induced alpha-helix formation of melittin are identical to those observed earlier for other peptides with an enthalpy h(helix) of -0.7 kcal/mol and a free energy g(helix) of -0.2 kcal/mol per peptide residue. These thermodynamic parameters hence appear to be of general validity for lipid-induced membrane folding. As g(helix) is negative, it further follows that helix formation leads to an enhanced membrane binding for the peptides or proteins involved. In this study, melittin binds by approximately 2 orders of magnitude better to the lipid membrane than [D]-V(5,8),I(17),K(21)-melittin which cannot form an alpha-helix. We also found conditions under which the isothermal titration experiment reports only the aggregation process. Melittin aggregation is an entropy-driven process with an endothermic heat of reaction (DeltaH(agg)) of approximately 2 kcal/mol and an aggregation constant of 20-40 M(-1).

Membrane Curvature and Lipid Composition Synergize To Regulate N-Ras Anchor Recruitment.

PubMed

Larsen, Jannik B; Kennard, Celeste; Pedersen, Søren L; Jensen, Knud J; Uline, Mark J; Hatzakis, Nikos S; Stamou, Dimitrios

2017-09-19

Proteins anchored to membranes through covalently linked fatty acids and/or isoprenoid groups play crucial roles in all forms of life. Sorting and trafficking of lipidated proteins has traditionally been discussed in the context of partitioning to membrane domains of different lipid composition. We recently showed that membrane shape/curvature can in itself mediate the recruitment of lipidated proteins. However, exactly how membrane curvature and composition synergize remains largely unexplored. Here we investigated how three critical structural parameters of lipids, namely acyl chain saturation, headgroup size, and acyl chain length, modulate the capacity of membrane curvature to recruit lipidated proteins. As a model system we used the lipidated minimal membrane anchor of the GTPase, N-Ras (tN-Ras). Our data revealed complex synergistic effects, whereby tN-Ras binding was higher on planar DOPC than POPC membranes, but inversely higher on curved POPC than DOPC membranes. This variation in the binding to both planar and curved membranes leads to a net increase in the recruitment by membrane curvature of tN-Ras when reducing the acyl chain saturation state. Additionally, we found increased recruitment by membrane curvature of tN-Ras when substituting PC for PE, and when decreasing acyl chain length from 14 to 12 carbons (DMPC versus DLPC). However, these variations in recruitment ability had different origins, with the headgroup size primarily influencing tN-Ras binding to planar membranes whereas the change in acyl chain length primarily affected binding to curved membranes. Molecular field theory calculations recapitulated these findings and revealed lateral pressure as an underlying biophysical mechanism dictating how curvature and composition synergize to modulate recruitment of lipidated proteins. Our findings suggest that the different compositions of cellular compartments could modulate the potency of membrane curvature to recruit lipidated proteins and thereby synergistically regulate the trafficking and sorting of lipidated proteins. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Effects of rare earth elements and REE-binding proteins on physiological responses in plants.

PubMed

Liu, Dongwu; Wang, Xue; Chen, Zhiwei

2012-02-01

Rare earth elements (REEs), which include 17 elements in the periodic table, share chemical properties related to a similar external electronic configuration. REEs enriched fertilizers have been used in China since the 1980s. REEs could enter the cell and cell organelles, influence plant growth, and mainly be bound with the biological macromolecules. REE-binding proteins have been found in some plants. In addition, the chlorophyll activities and photosynthetic rate can be regulated by REEs. REEs could promote the protective function of cell membrane and enhance the plant resistance capability to stress produced by environmental factors, and affect the plant physiological mechanism by regulating the Ca²⁺ level in the plant cells. The focus of present review is to describe how REEs and REE-binding proteins participate in the physiological responses in plants.

Crystal structure of a catalytically active, non-toxic endopeptidase derivative of Clostridium botulinum toxin A.

PubMed

Masuyer, Geoffrey; Thiyagarajan, Nethaji; James, Peter L; Marks, Philip M H; Chaddock, John A; Acharya, K Ravi

2009-03-27

Botulinum neurotoxins (BoNTs) modulate cholinergic nerve terminals to result in neurotransmitter blockade. BoNTs consists of catalytic (LC), translocation (Hn) and cell-binding domains (Hc). The binding function of the Hc domain is essential for BoNTs to bind the neuronal cell membrane, therefore, removal of the Hc domain results in a product that retains the endopeptidase activity of the LC but is non-toxic. Thus, a molecule consisting of LC and Hn domains of BoNTs, termed LHn, is a suitable molecule for engineering novel therapeutics. The structure of LHA at 2.6 A reported here provides an understanding of the structural implications and challenges of engineering therapeutic molecules that combine functional properties of LHn of BoNTs with specific ligand partners to target different cell types.

Self-assembled tethered bimolecular lipid membranes.

PubMed

Sinner, Eva-Kathrin; Ritz, Sandra; Naumann, Renate; Schiller, Stefan; Knoll, Wolfgang

2009-01-01

This chapter describes some of the strategies developed in our group for designing, constructing and structurally and functionally characterizing tethered bimolecular lipid membranes (tBLM). We introduce this platform as a novel model membrane system that complements the existing ones, for example, Langmuir monolayers, vesicular liposomal dispersions and bimolecular ("black") lipid membranes. Moreover, it offers the additional advantage of allowing for studies of the influence of membrane structure and order on the function of integral proteins, for example, on how the composition and organization of lipids in a mixed membrane influence the ion translocation activity of integral channel proteins. The first strategy that we introduce concerns the preparation of tethered monolayers by the self-assembly of telechelics. Their molecular architecture with a headgroup, a spacer unit (the "tether") and the amphiphile that mimics the lipid molecule allows them to bind specifically to the solid support thus forming the proximal layer of the final architecture. After fusion of vesicles that could contain reconstituted proteins from a liposomal dispersion in contact to this monolayer the tethered bimolecular lipid membrane is obtained. This can then be characterized by a broad range of surface analytical techniques, including surface plasmon spectroscopies, the quartz crystal microbalance, fluorescence and IR spectroscopies, and electrochemical techniques, to mention a few. It is shown that this concept allows for the construction of tethered lipid bilayers with outstanding electrical properties including resistivities in excess of 10 MOmega cm2. A modified strategy uses the assembly of peptides as spacers that couple covalently via their engineered sulfhydryl or lipoic acid groups at the N-terminus to the employed gold substrate, while their C-terminus is being activated afterward for the coupling of, for example, dimyristoylphosphatidylethanol amine (DMPE) lipid molecules via the NH2 moiety of their headgroups. It is demonstrated that these membranes are well suited for the in situ synthesis of membrane protein by a cell-free expression approach. The vectorial integration of an in vitro synthesized odorant receptor, OR5 from the rat, is demonstrated by means of antibodies that specifically bind to a tag at the N-terminus of the receptor and is read out by surface plasmon fluorescence spectroscopy. A completely different strategy employs his-tagged membrane proteins in their solubilized form binding to a surface-attached Ni(+)-NTA monolayer generating a well-oriented protein layer the density of which can be easily controlled by online monitoring the binding (assembly) step by surface plasmon spectroscopy. Moreover, the attachment of the his-tag to either the C- or the N-terminus allows for the complete control of the protein orientation. After the exchange of the detergent micelle by a lipid bilayer via a surface dialysis procedure an electrically very well isolating protein-tethered membrane is formed. We show that this "wiring" of the functional units allows for the (external) manipulation of the oxidation state of the redox-protein cytochrome c Oxidase by the control of the potential applied to the gold substrate which is used as the working electrode in an electrochemical attachment.

Strychnine Binding Associated with Glycine Receptors of the Central Nervous System

PubMed Central

Young, Anne B.; Snyder, Solomon H.

1973-01-01

[3H]Strychnine binds to synaptic-membrane fractions of the spinal cord in a selective fashion, indicating an interaction with postsynaptic glycine receptors. Displacement of strychnine by glycine and other amino acids parallels their glycine-like neurophysiologic activity. The regional localization of strychnine binding in the central nervous system correlates closely with endogenous glycine concentrations. In subcellular fractionation experiments, strychnine binding is most enhanced in synaptic-membrane fractions. Strychnine binding is saturable, with affinity constants for glycine and strychnine of 10 and 0.03 μM, respectively. PMID:4200724

Bile acids modulate signaling by functional perturbation of plasma membrane domains.

PubMed

Zhou, Yong; Maxwell, Kelsey N; Sezgin, Erdinc; Lu, Maryia; Liang, Hong; Hancock, John F; Dial, Elizabeth J; Lichtenberger, Lenard M; Levental, Ilya

2013-12-13

Eukaryotic cell membranes are organized into functional lipid and protein domains, the most widely studied being membrane rafts. Although rafts have been associated with numerous plasma membrane functions, the mechanisms by which these domains themselves are regulated remain undefined. Bile acids (BAs), whose primary function is the solubilization of dietary lipids for digestion and absorption, can affect cells by interacting directly with membranes. To investigate whether these interactions affected domain organization in biological membranes, we assayed the effects of BAs on biomimetic synthetic liposomes, isolated plasma membranes, and live cells. At cytotoxic concentrations, BAs dissolved synthetic and cell-derived membranes and disrupted live cell plasma membranes, implicating plasma membrane damage as the mechanism for BA cellular toxicity. At subtoxic concentrations, BAs dramatically stabilized domain separation in Giant Plasma Membrane Vesicles without affecting protein partitioning between coexisting domains. Domain stabilization was the result of BA binding to and disordering the nonraft domain, thus promoting separation by enhancing domain immiscibility. Consistent with the physical changes observed in synthetic and isolated biological membranes, BAs reorganized intact cell membranes, as evaluated by the spatial distribution of membrane-anchored Ras isoforms. Nanoclustering of K-Ras, related to nonraft membrane domains, was enhanced in intact plasma membranes, whereas the organization of H-Ras was unaffected. BA-induced changes in Ras lateral segregation potentiated EGF-induced signaling through MAPK, confirming the ability of BAs to influence cell signal transduction by altering the physical properties of the plasma membrane. These observations suggest general, membrane-mediated mechanisms by which biological amphiphiles can produce their cellular effects.

Molecular basis of endosomal-membrane association for the dengue virus envelope protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rogers, David M.; Kent, Michael S.; Rempe, Susan B.

Dengue virus is coated by an icosahedral shell of 90 envelope protein dimers that convert to trimers at low pH and promote fusion of its membrane with the membrane of the host endosome. We provide the first estimates for the free energy barrier and minimum for two key steps in this process: host membrane bending and protein–membrane binding. Both are studied using complementary membrane elastic, continuum electrostatics and all-atom molecular dynamics simulations. The predicted host membrane bending required to form an initial fusion stalk presents a 22–30 kcal/mol free energy barrier according to a constrained membrane elastic model. Combined continuummore » and molecular dynamics results predict a 15 kcal/mol free energy decrease on binding of each trimer of dengue envelope protein to a membrane with 30% anionic phosphatidylglycerol lipid. The bending cost depends on the preferred curvature of the lipids composing the host membrane leaflets, while the free energy gained for protein binding depends on the surface charge density of the host membrane. The fusion loop of the envelope protein inserts exactly at the level of the interface between the membrane's hydrophobic and head-group regions. As a result, the methods used in this work provide a means for further characterization of the structures and free energies of protein-assisted membrane fusion.« less

Molecular basis of endosomal-membrane association for the dengue virus envelope protein

DOE PAGES

Rogers, David M.; Kent, Michael S.; Rempe, Susan B.

2015-01-02

Dengue virus is coated by an icosahedral shell of 90 envelope protein dimers that convert to trimers at low pH and promote fusion of its membrane with the membrane of the host endosome. We provide the first estimates for the free energy barrier and minimum for two key steps in this process: host membrane bending and protein–membrane binding. Both are studied using complementary membrane elastic, continuum electrostatics and all-atom molecular dynamics simulations. The predicted host membrane bending required to form an initial fusion stalk presents a 22–30 kcal/mol free energy barrier according to a constrained membrane elastic model. Combined continuummore » and molecular dynamics results predict a 15 kcal/mol free energy decrease on binding of each trimer of dengue envelope protein to a membrane with 30% anionic phosphatidylglycerol lipid. The bending cost depends on the preferred curvature of the lipids composing the host membrane leaflets, while the free energy gained for protein binding depends on the surface charge density of the host membrane. The fusion loop of the envelope protein inserts exactly at the level of the interface between the membrane's hydrophobic and head-group regions. As a result, the methods used in this work provide a means for further characterization of the structures and free energies of protein-assisted membrane fusion.« less

Concentration of isoprene in artificial and thylakoid membranes.

PubMed

Harvey, Christopher M; Li, Ziru; Tjellström, Henrik; Blanchard, Gary J; Sharkey, Thomas D

2015-10-01

Isoprene emission protects plants from a variety of abiotic stresses. It has been hypothesized to do so by partitioning into cellular membranes, particularly the thylakoid membrane. At sufficiently high concentrations, this partitioning may alter the physical properties of membranes. As much as several per cent of carbon taken up in photosynthesis is re-emitted as isoprene but the concentration of isoprene in the thylakoid membrane of rapidly emitting plants has seldom been considered. In this study, the intramembrane concentration of isoprene in phosphatidylcholine liposomes equilibrated to a physiologically relevant gas phase concentration of 20 μL L(-1) isoprene was less than predicted by ab initio calculations based on the octanol-water partitioning coefficient of isoprene while the concentration in thylakoid membranes was more. However, the concentration in both systems was roughly two orders of magnitude lower than previously assumed. High concentrations of isoprene (2000 μL L(-1) gas phase) failed to alter the viscosity of phosphatidylcholine liposomes as measured with perylene, a molecular probe of membrane structure. These results strongly suggest that the physiological concentration of isoprene within the leaves of highly emitting plants is too low to affect the dynamics of thylakoid membrane acyl lipids. It is speculated that isoprene may bind to and modulate the dynamics of thylakoid embedded proteins.

Interaction of MreB-derived antimicrobial peptides with membranes.

PubMed

Saikia, Karabi; Chaudhary, Nitin

2018-03-25

Antimicrobial peptides are critical components of defense systems in living forms. The activity is conferred largely by the selective membrane-permeabilizing ability. In our earlier work, we derived potent antimicrobial peptides from the 9-residue long, N-terminal amphipathic helix of E. coli MreB protein. The peptides display broad-spectrum activity, killing not only Gram-positive and Gram-negative bacteria but opportunistic fungus, Candida albicans as well. These results proved that membrane-binding stretches of bacterial proteins could turn out to be self-harming when applied from outside. Here, we studied the membrane-binding and membrane-perturbing potential of these peptides. Steady-state tryptophan fluorescence studies with tryptophan extended peptides, WMreB 1-9 and its N-terminal acetylated analog, Ac-WMreB 1-9 show preferential binding to negatively-charged liposomes. Both the peptides cause permeabilization of E. coli inner and outer-membranes. Tryptophan-lacking peptides, though permeabilize the outer-membrane efficiently, little permeabilization of the inner-membrane is observed. These data attest membrane-destabilization as the mechanism of rapid bacterial killing. This study is expected to motivate the research in identifying microbes' self-sequences to combat them. Copyright © 2018 Elsevier Inc. All rights reserved.

Biotechnologically relevant enzymes and proteins. Antifungal mechanism of the Aspergillus giganteus AFP against the rice blast fungus Magnaporthe grisea.

PubMed

Moreno, Ana Beatriz; Martínez Del Pozo, Alvaro; San Segundo, Blanca

2006-10-01

The mold Aspergillus giganteus produces a basic, low molecular weight protein showing antifungal properties against economically important plant pathogens, the AFP (Antifungal Protein). In this study, we investigated the mechanisms by which AFP exerts its antifungal activity against Magnaporthe grisea. M. grisea is the causal agent of rice blast, one of the most devastating diseases of cultivated rice worldwide. AFP was purified from the extracellular medium of A. giganteus cultures. The AFP protein was found to induce membrane permeabilization in M. grisea cells. Electron microscopy studies revealed severe cellular degradation and damage of plasma membranes in AFP-treated fungal cells. AFP however failed to induce membrane permeabilization on rice or human HeLa cells. Furthermore, AFP enters the fungal cell and targets to the nucleus, as revealed by co-localization experiments of Alexa-labeled AFP with the SYTOX Green dye. Finally, AFP binds to nucleic acids, including M. grisea DNA. Our results suggest that the combination of fungal cell permeabilization, cell-penetrating ability and nucleic acid-binding activity of AFP determines its potent antifungal activity against M. grisea. These results are discussed in relation to the potential of the AFP protein to enhance crop protection against fungal diseases.

Localization of Bacillus thuringiensis Cry1A toxin-binding molecules in gypsy moth larval gut sections using fluorescence microscopy

Treesearch

Algimantas P. Valaitis

2011-01-01

The microbial insecticide Bacillus thuringiensis (Bt) produces Cry toxins, proteins that bind to the brush border membranes of gut epithelial cells of insects that ingest it, disrupting the integrity of the membranes, and leading to cell lysis and insect death. In gypsy moth, Lymantria dispar, two toxin-binding molecules for the...

Lactose carrier protein of Escherichia coli. Transport and binding of 2'-(N-dansyl)aminoethyl beta-D-thiogalactopyranoside and p-nitrophenyl alpha-d-galactopyranoside.

PubMed

Overath, P; Teather, R M; Simoni, R D; Aichele, G; Wilhelm, U

1979-01-09

The elevated level of lactose carrier protein present in cytoplasmic membranes derived from Escherichia coli strain T31RT, which carries the Y gene of the lac operon on a plasmid vector (Teather, R. M., et al. (1978) Mol. Gen. Genet. 159, 239--248), has allowed the detection of a complex between the carrier and the fluorescent substrate 2'-(N-dansyl)-aminoethyl beta-D-thiogalactopyranoside (Dns2-S-Gal). Binding is accompanied by a 50-nm blue shift in the emission maximum of the dansyl residue. The complex (dissociation constant, KD = 30 micron) rapidly dissociates upon addition of competing substrates such as beta-D-galactopyranosyl 1-thio-beta-D-galactopyranoside or upon reaction with the thiol reagent p-chloromercuribenzenesulfonate. Binding of both Dns2-S-Gal and p-nitrophenyl alpha-D-galactopyranoside (alpha-NPG) occurs spontaneously in the absence of an electrochemical potential gradient across the membrane. Comparison of equilibrium binding experiments using Dns2-S-Gal or alpha-NPG and differential labeling of the carrier with radioactive amino acids shows that the carrier binds 1 mol of substrate per mol of polypeptide (molecular weight 30 000). In addition to specific binding to the lactose carrier, Dns2-S-gal binds unspecifically to lipid vesicles or membranes, as described by a partition coefficient, K = 60, resulting in a 25-nm blue shift in the emission maximum of the dansyl group. Both Dns2-S-Gal and alpha-NPG are not only bound by the lactose carrier but also transported across the membrane by this transport protein in cells and membrane vesicles. The fluorescence changes observed with dansylated galactosides in membrane vesicles in the presence of an electrochemical gradient (Schuldiner et al. (1975) J. Biol. Chem. 250, 1361--1370)) are interpreted as an increase in unspecific binding after translocation.

MinD-dependent conformational changes in MinE required for the Min oscillator to spatially regulate cytokinesis

PubMed Central

Park, Kyung-Tae; Wu, Wei; Battaile, Kevin P.; Lovell, Scott; Holyoak, Todd; Lutkenhaus, Joe

2011-01-01

Summary MinD recruits MinE to the membrane leading to a coupled oscillation required for spatial regulation of the cytokinetic Z ring in E. coli. How these proteins interact, however, is not clear since the MinD binding regions of MinE are sequestered within a 6-stranded β-sheet and masked by N-terminal helices. Here, minE mutations are isolated that restore interaction to some MinD and MinE mutants. These mutations alter the MinE structure releasing the MinD binding regions and N-terminal helices that bind MinD and the membrane, respectively. Crystallization of MinD-MinE complexes reveals a 4-stranded β-sheet MinE dimer with the released β strands (MinD binding regions) converted to α-helices bound to MinD dimers. These results suggest a 6 stranded, β-sheet dimer of MinE ‘senses’ MinD and switches to a 4-stranded β-sheet dimer that binds MinD and contributes to membrane binding. Also, the results indicate how MinE persists at the MinD-membrane surface. PMID:21816275

Heterotypic binding between neuronal membrane vesicles and glial cells is mediated by a specific cell adhesion molecule

PubMed Central

1984-01-01

By means of a multistage quantitative assay, we have identified a new kind of cell adhesion molecule (CAM) on neuronal cells of the chick embryo that is involved in their adhesion to glial cells. The assay used to identify the binding component (which we name neuron-glia CAM or Ng-CAM) was designed to distinguish between homotypic binding (e.g., neuron to neuron) and heterotypic binding (e.g., neuron to glia). This distinction was essential because a single neuron might simultaneously carry different CAMs separately mediating each of these interactions. The adhesion of neuronal cells to glial cells in vitro was previously found to be inhibited by Fab' fragments prepared from antisera against neuronal membranes but not by Fab' fragments against N-CAM, the neural cell adhesion molecule. This suggested that neuron-glia adhesion is mediated by specific cell surface molecules different from previously isolated CAMs . To verify that this was the case, neuronal membrane vesicles were labeled internally with 6-carboxyfluorescein and externally with 125I-labeled antibodies to N-CAM to block their homotypic binding. Labeled vesicles bound to glial cells but not to fibroblasts during a 30-min incubation period. The specific binding of the neuronal vesicles to glial cells was measured by fluorescence microscopy and gamma spectroscopy of the 125I label. Binding increased with increasing concentrations of both glial cells and neuronal vesicles. Fab' fragments prepared from anti-neuronal membrane sera that inhibited binding between neurons and glial cells were also found to inhibit neuronal vesicle binding to glial cells. The inhibitory activity of the Fab' fragments was depleted by preincubation with neuronal cells but not with glial cells. Trypsin treatment of neuronal membrane vesicles released material that neutralized Fab' fragment inhibition; after chromatography, neutralizing activity was enriched 50- fold. This fraction was injected into mice to produce monoclonal antibodies; an antibody was obtained that interacted with neurons, inhibited binding of neuronal membrane vesicles to glial cells, and recognized an Mr = 135,000 band in immunoblots of embryonic chick brain membranes. These results suggest that this molecule is present on the surfaces of neurons and that it directly or indirectly mediates adhesion between neurons and glial cells. Because the monoclonal antibody as well as the original polyspecific antibodies that were active in the assay did not bind to glial cells, we infer that neuron- glial interaction is heterophilic, i.e., it occurs between Ng-CAM on neurons and an as yet unidentified CAM present on glial cells. PMID:6725397

Microimaging of Bacillus thuringiensis Toxin-binding proteins in gypsy moth larval gut using confocal fluorescence microscopy

Treesearch

Daniel J. Krofcheck; Algimantas P. Valaitis

2010-01-01

After ingestion by susceptible insect larvae, Bacillus thuringiensis (Bt) insecticidal proteins bind to the brush border membranes of gut epithelial cells and disrupt the integrity of the plasma membrane by forming...

Mutagenesis of Paramyxovirus Hemagglutinin-Neuraminidase Membrane-Proximal Stalk Region Influences Stability, Receptor Binding, and Neuraminidase Activity.

PubMed

Adu-Gyamfi, Emmanuel; Kim, Lori S; Jardetzky, Theodore S; Lamb, Robert A

2016-09-01

Paramyxoviridae consist of a large family of enveloped, negative-sense, nonsegmented single-stranded RNA viruses that account for a significant number of human and animal diseases. The fusion process for nearly all paramyxoviruses involves the mixing of the host cell plasma membrane and the virus envelope in a pH-independent fashion. Fusion is orchestrated via the concerted action of two surface glycoproteins: an attachment protein called hemagglutinin-neuraminidase (HN [also called H or G depending on virus type and substrate]), which acts as a receptor binding protein, and a fusion (F) protein, which undergoes a major irreversible refolding process to merge the two membranes. Recent biochemical evidence suggests that receptor binding by HN is dispensable for cell-cell fusion. However, factors that influence the stability and/or conformation of the HN 4-helix bundle (4HB) stalk have not been studied. Here, we used oxidative cross-linking as well as functional assays to investigate the role of the structurally unresolved membrane-proximal stalk region (MPSR) (residues 37 to 58) of HN in the context of headless and full-length HN membrane fusion promotion. Our data suggest that the receptor binding head serves to stabilize the stalk to regulate fusion. Moreover, we found that the MPSR of HN modulates receptor binding and neuraminidase activity without a corresponding regulation of F triggering. Paramyxoviruses require two viral membrane glycoproteins, the attachment protein variously called HN, H, or G and the fusion protein (F), to couple host receptor recognition to virus-cell fusion. The HN protein has a globular head that is attached to a membrane-anchored flexible stalk of ∼80 residues and has three activities: receptor binding, neuraminidase, and fusion activation. In this report, we have identified the functional significance of the membrane-proximal stalk region (MPSR) (HN, residues 37 to 56) of the paramyxovirus parainfluenza virus (PIV5), a region of the HN stalk that has not had its structure determined by X-ray crystallography. Our data suggest that the MPSR influences receptor binding and neuraminidase activity via an indirect mechanism. Moreover, the receptor binding head group stabilizes the 4HB stalk as part of the general mechanism to fine-tune F-activation. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Binding of Thyrotropin-Releasing Hormone to Plasma Membranes of Bovine Anterior Pituitary Gland

PubMed Central

Labrie, Fernand; Barden, Nicholas; Poirier, Guy; De Lean, Andre

1972-01-01

An assay for the binding of [3H]thyrotropin-releasing hormone ([3H]TRH) is described. Plasma membranes isolated from bovine anterior pituitary gland bind about 600 femtomoles of this hormone per mg of protein, as compared to 15 femtomoles per mg of protein in the total adenohypophyseal homogenate (40-fold purification). The equilibrium constant of membrane receptor-[3H]TRH binding at 0°C is 4.3 × 107 L·M-1, or a half-maximal binding of this hormone at 23 nM. The binding is time-dependent; addition of unlabeled hormone induces dissociation of the receptor-[3H]TRH complex with a half-life of 14 min. The binding of TRH is not altered by 10 μM melanocyte-stimulating hormone-release inhibiting hormone, lysine-vasopressin, adrenocorticotropin, growth hormone, prolactin, luteinizing hormone, insulin, glucagon, L-thyroxine, or L-triiodothyronine. K+ and Mg++ increase formation of the receptor-TRH complex at optimal concentrations of 5-25 mM and 0.5-2.5 mM, respectively, with inhibition at higher concentrations. Ca++ inhibits binding of TRH at all concentrations tested. PMID:4621548

Interaction of Gramicidin S and its Aromatic Amino-Acid Analog with Phospholipid Membranes

PubMed Central

Jelokhani-Niaraki, Masoud; Hodges, Robert S.; Meissner, Joseph E.; Hassenstein, Una E.; Wheaton, Laura

2008-01-01

To investigate the mechanism of interaction of gramicidin S-like antimicrobial peptides with biological membranes, a series of five decameric cyclic cationic β-sheet-β-turn peptides with all possible combinations of aromatic D-amino acids, Cyclo(Val-Lys-Leu-D-Ar1-Pro-Val-Lys-Leu-D-Ar2-Pro) (Ar ≡ Phe, Tyr, Trp), were synthesized. Conformations of these cyclic peptides were comparable in aqueous solutions and lipid vesicles. Isothermal titration calorimetry measurements revealed entropy-driven binding of cyclic peptides to POPC and POPE/POPG lipid vesicles. Binding of peptides to both vesicle systems was endothermic—exceptions were peptides containing the Trp-Trp and Tyr-Trp pairs with exothermic binding to POPC vesicles. Application of one- and two-site binding (partitioning) models to binding isotherms of exothermic and endothermic binding processes, respectively, resulted in determination of peptide-lipid membrane binding constants (Kb). The Kb1 and Kb2 values for endothermic two-step binding processes corresponded to high and low binding affinities (Kb1 ≥ 100 Kb2). Conformational change of cyclic peptides in transferring from buffer to lipid bilayer surfaces was estimated using fluorescence resonance energy transfer between the Tyr-Trp pair in one of the peptide constructs. The cyclic peptide conformation expands upon adsorption on lipid bilayer surface and interacts more deeply with the outer monolayer causing bilayer deformation, which may lead to formation of nonspecific transient peptide-lipid porelike zones causing membrane lysis. PMID:18621820

Activation mechanism of erythrocyte cathepsin E. evidence for the occurrence of the membrane-associated active enzyme.

PubMed

Ueno, E; Sakai, H; Kato, Y; Yamamoto, K

1989-06-01

Activation of the erythrocyte cathepsin E located on the cytoplasmic surface of the membrane in a latent form was studied in stripped inside-out membrane vesicles prepared from human erythrocyte membranes. Incubation of the vesicles at 40 degrees C at pH 4 resulted in increased degradation of the membrane proteins, especially band 3. This proteolysis was selectively inhibited by the inclusion of pepstatin (isovaleryl-Val-Val-statyl-Ala-statine) or H 297 [Pro-Thr-Glu-Phe(CH2-NH)Nle-Arg-Leu] in the incubation mixtures, indicating that cathepsin E, as the only aspartic proteinase in erythrocytes, is responsible for the proteolysis. Two potential active-site-directed inhibitors of aspartic proteinases, pepstatin and H 297, were used to prove the occurrence of the membrane-associated active enzyme. To minimize potential errors arising from non-specific binding, the concentrations of the inhibitors used in the binding assay (pepstatin, 5 x 10(-8) M; H 297, 1 x 10(-5) M) were determined by calibration for purified and membrane-associated cathepsin E. The inhibition of the membrane-associated cathepsin E by each inhibitor, which showed the binding of the inhibitor to the activated enzyme, was temperature- and time-dependent. The binding of each inhibitor to the enzyme on the exposed surface of the membrane at pH 4 was highly specific, saturable, and reversible. The present study thus provides the first evidence that cathepsin E tightly bound to the membrane is converted to the active enzyme in the membrane-associated form, and suggests that this enzyme may be responsible for the degradation of band 3.

Role of Annular Lipids in the Functional Properties of Leucine Transporter LeuT Proteomicelles.

PubMed

LeVine, Michael V; Khelashvili, George; Shi, Lei; Quick, Matthias; Javitch, Jonathan A; Weinstein, Harel

2016-02-16

Recent work has shown that the choice of the type and concentration of detergent used for the solubilization of membrane proteins can strongly influence the results of functional experiments. In particular, the amino acid transporter LeuT can bind two substrate molecules in low concentrations of n-dodecyl β-d-maltopyranoside (DDM), whereas high concentrations reduce the molar binding stoichiometry to 1:1. Subsequent molecular dynamics (MD) simulations of LeuT in DDM proteomicelles revealed that DDM can penetrate to the extracellular vestibule and make stable contacts in the functionally important secondary substrate binding site (S2), suggesting a potential competitive mechanism for the reduction in binding stoichiometry. Because annular lipids can be retained during solubilization, we performed MD simulations of LeuT proteomicelles at various stages of the solubilization process. We find that at low DDM concentrations, lipids are retained around the protein and penetration of detergent into the S2 site does not occur, whereas at high concentrations, lipids are displaced and the probability of DDM binding in the S2 site is increased. This behavior is dependent on the type of detergent, however, as we find in the simulations that the detergent lauryl maltose-neopentyl glycol, which is approximately twice the size of DDM and structurally more closely resembles lipids, does not penetrate the protein even at very high concentrations. We present functional studies that confirm the computational findings, emphasizing the need for careful consideration of experimental conditions, and for cautious interpretation of data in gathering mechanistic information about membrane proteins.

Role of Annular Lipids in the Functional Properties of Leucine Transporter LeuT Proteomicelles

PubMed Central

2016-01-01

Recent work has shown that the choice of the type and concentration of detergent used for the solubilization of membrane proteins can strongly influence the results of functional experiments. In particular, the amino acid transporter LeuT can bind two substrate molecules in low concentrations of n-dodecyl β-d-maltopyranoside (DDM), whereas high concentrations reduce the molar binding stoichiometry to 1:1. Subsequent molecular dynamics (MD) simulations of LeuT in DDM proteomicelles revealed that DDM can penetrate to the extracellular vestibule and make stable contacts in the functionally important secondary substrate binding site (S2), suggesting a potential competitive mechanism for the reduction in binding stoichiometry. Because annular lipids can be retained during solubilization, we performed MD simulations of LeuT proteomicelles at various stages of the solubilization process. We find that at low DDM concentrations, lipids are retained around the protein and penetration of detergent into the S2 site does not occur, whereas at high concentrations, lipids are displaced and the probability of DDM binding in the S2 site is increased. This behavior is dependent on the type of detergent, however, as we find in the simulations that the detergent lauryl maltose-neopentyl glycol, which is approximately twice the size of DDM and structurally more closely resembles lipids, does not penetrate the protein even at very high concentrations. We present functional studies that confirm the computational findings, emphasizing the need for careful consideration of experimental conditions, and for cautious interpretation of data in gathering mechanistic information about membrane proteins. PMID:26811944

Selective Interactions of Valeriana officinalis Extracts and Valerenic Acid with [H]Glutamate Binding to Rat Synaptic Membranes.

PubMed

Del Valle-Mojica, Lisa M; Ayala-Marín, Yoshira M; Ortiz-Sanchez, Carmen M; Torres-Hernández, Bianca A; Abdalla-Mukhaimer, Safa; Ortiz, José G

2011-01-01

Although GABA neurotransmission has been suggested as a mechanism for Valeriana officinalis effects, CNS depression can also be evoked by inhibition of ionotropic (iGluR) and metabotropic glutamate receptors (mGluR). In this study, we examined if aqueous valerian extract interacted with glutamatergic receptors. Freshly prepared aqueous valerian extract was incubated with rat cortical synaptic membranes in presence of 20 nM [(3)H]Glutamate. Aqueous valerian extract increased [(3)H]Glutamate binding from 1 × 10(-7) to 1 × 10(-3) mg/mL. In the presence of (2S,1'S,2'S)-2-(Carboxycyclopropyl)glycine (LCCG-I) and (2S,2'R,3'R)-2-(2',3'-Dicarboxycyclopropyl)glycine (DCG-IV), Group II mGluR agents, valerian extract markedly decreased [(3)H]Glutamate binding, while (2S)-2-amino-3-(3,5-dioxo-1,2,4-oxadiazolidin-2-yl) propanoic acid) (quisqualic acid, QA), Group I mGluR agonist, increased [(3)H]Glutamate binding. At 0.05 mg/mL aqueous valerian extract specifically interacted with kainic acid NMDA and AMPA receptors. Valerenic acid, a marker compound for Valeriana officinalis, increased the [(3)H]Glutamate binding after 1.6 × 10(-2) mg/mL, and at 0.008 mg/mL it interacted only with QA (Group I mGluR). The selective interactions of valerian extract and valerenic acid with Group I and Group II mGluR may represent an alternative explanation for the anxiolytic properties of this plant.

Modular Synthesis of Biologically Active Phosphatidic Acid Probes Using Click Chemistry

PubMed Central

Smith, Matthew D.; Sudhahar, Christopher G.; Gong, Denghuang; Stahelin, Robert V.

2018-01-01

Phosphatidic acid (PA) is an important signaling lipid that plays roles in a range of biological processes including both physiological and pathophysiological events. PA is one of a number of signaling lipids that can act as site-specific ligands for protein receptors in binding events that enforce membrane-association and generally regulate both receptor function and subcellular localization. However, elucidation of the full scope of PA activities has proven problematic, primarily due to the lack of a consensus sequence among PA-binding receptors. Thus, experimental approaches, such as those employing lipid probes, are necessary for characterizing interactions at the molecular level. Herein, we describe an efficient modular approach to the synthesis of a range of PA probes that employs a late stage introduction of reporter groups. This strategy was exploited in the synthesis of PA probes bearing fluorescent and photoaffinity tags as well as a bifunctional probe containing both a photoaffinity moiety and an azide as a secondary handle for purification purposes. To discern the ability of these PA analogues to mimic the natural lipid in protein binding properties, each compound was incorporated into vesicles for binding studies using a known PA receptor, the C2 domain of PKCα. In these studies, each compound exhibited binding properties that were comparable to those of synthetic PA, indicating their viability as probes for effectively studying the activities of PA in cellular processes. PMID:19668861

G-protein mediates voltage regulation of agonist binding to muscarinic receptors: effects on receptor-Na/sup +/ channel interaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cohen-Armon, M.; Garty, H.; Sokolovsky, M.

1988-01-12

The authors previous experiments in membranes prepared from rat heart and brain led them to suggest that the binding of agonist to the muscarinic receptors and to the Na/sup +/ channels is a coupled event mediated by guanine nucleotide binding protein(s) (G-protein(s)). These in vitro findings prompted us to employ synaptoneurosomes from brain stem tissue to examine (i) the binding properties of (/sup 3/H) acetylcholine at resting potential and under depolarization conditions in the absence and presence of pertussis toxin; (ii) the binding of (/sup 3/H)batrachotoxin to Na/sup +/ channel(s) in the presence of the muscarinic agonists; and (iii) muscarinicallymore » induced /sup 22/Na/sup +/ uptake in the presence and absence of tetrodotoxin, which blocks Na/sup +/ channels. The findings indicate that agonist binding to muscarinic receptors is voltage dependent, that this process is mediated by G-protein(s), and that muscarinic agonists induce opening of Na/sup +/channels. The latter process persists even after pertussis toxin treatment, indicating that it is not likely to be mediated by pertussis toxin sensitive G-protein(s). The system with its three interacting components-receptor, G-protein, and Na/sup +/ channel-is such that at resting potential the muscarinic receptor induces opening of Na/sup +/ channels; this property may provide a possible physiological mechanism for the depolarization stimulus necessary for autoexcitation or repetitive firing in heart or brain tissues.« less

Ankyrin binding activity shared by the neurofascin/L1/NrCAM family of nervous system cell adhesion molecules.

PubMed

Davis, J Q; Bennett, V

1994-11-04

Neurofascin, L1, NrCAM, NgCAM, and neuroglian are membrane-spanning cell adhesion molecules with conserved cytoplasmic domains that are believed to play important roles in development of the nervous system. This report presents biochemical evidence that the cytoplasmic domains of these molecules associate directly with ankyrins, a family of spectrin-binding proteins located on the cytoplasmic surface of specialized plasma membrane domains. Rat neurofascin and NrCAM together comprise over 0.5% of the membrane protein in adult brain tissue. Linkage of these ankyrin-binding cell adhesion molecules to spectrin-based structures may provide a major class of membrane-cytoskeletal connections in adult brain as well as earlier stages of development.

Human EAG channels are directly modulated by PIP2 as revealed by electrophysiological and optical interference investigations

PubMed Central

Han, Bo; He, Kunyan; Cai, Chunlin; Tang, Yin; Yang, Linli; Heinemann, Stefan H.; Hoshi, Toshinori; Hou, Shangwei

2016-01-01

Voltage-gated ether à go-go (EAG) K+ channels are expressed in various types of cancer cells and also in the central nervous system. Aberrant overactivation of human EAG1 (hEAG1) channels is associated with cancer and neuronal disorders such as Zimmermann-Laband and Temple-Baraitser syndromes. Although hEAG1 channels are recognized as potential therapeutic targets, regulation of their functional properties is only poorly understood. Here, we show that the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) is a potent inhibitory gating modifier of hEAG1 channels. PIP2 inhibits the channel activity by directly binding to a short N-terminal segment of the channel important for Ca2+/calmodulin (CaM) binding as evidenced by bio-layer interferometry measurements. Conversely, depletion of endogenous PIP2 either by serotonin-induced phospholipase C (PLC) activation or by a rapamycin-induced translocation system enhances the channel activity at physiological membrane potentials, suggesting that PIP2 exerts a tonic inhibitory influence. Our study, combining electrophysiological and direct binding assays, demonstrates that hEAG1 channels are subject to potent inhibitory modulation by multiple phospholipids and suggests that manipulations of the PIP2 signaling pathway may represent a strategy to treat hEAG1 channel-associated diseases. PMID:27005320

Co-autodisplay of Z-domains and bovine caseins on the outer membrane of E. coli.

PubMed

Yoo, Gu; Saenger, Thorsten; Bong, Ji-Hong; Jose, Joachim; Kang, Min-Jung; Pyun, Jae-Chul

2015-12-01

In this work, two proteins, Z-domains and bovine casein, were auto-displayed on the outer membrane of the same Escherichia coli cells by co-transformation of two different auto-display vectors. On the basis of SDS-PAGE densitometry, Z-domains and bovine casein were expressed at 3.12 × 10⁵ and 1.55 × 10⁵ proteins/E. coli cell, respectively. The co-auto-displayed Z-domains had antibody-binding activity and the bovine casein had adhesive properties. E. coli with co-auto-displayed proteins were analyzed by fluorescence assisted cell sorting (FACS). E. coli with co-auto-displayed Z-domains and bovine casein aggregated due to hydrophobic interaction. For application to immunoassays, the Z-domain activity was estimated after (1) immobilizing the E. coli and (2) forming an OM layer. E. coli with co-auto-displayed two proteins that were immobilized on a polystyrene microplate had the same antibody-binding activity as did E. coli with auto-displayed Z-domains only. The OM layer from the co-transformed E. coli had Z-domains and bovine casein expressed at a 1:2 ratio from antibody-binding activity measurements. Copyright © 2015 Elsevier B.V. All rights reserved.

The interaction of substituted benzamides with brain benzodiazepine binding sites in vitro.

PubMed

Horton, R W; Lowther, S; Chivers, J; Jenner, P; Marsden, C D; Testa, B

1988-08-01

1. The interaction of substituted benzamides with brain benzodiazepine (BDZ) binding sites was examined by their ability to displace [3H]-flunitrazepam ([3H]-FNM) from specific binding sites in bovine cortical membranes in vitro. 2. Clebopride, Delagrange 2674, Delagrange 2335 and BRL 20627 displayed concentration-dependent displacement of [3H]-FNM with IC50 values of 73 nM, 132 nM, 7.7 microM and 5.9 microM, respectively. Other substituted benzamides including metoclopramide, sulpiride, tiapride, sultopride and cisapride were inactive at 10(-5) M. 3. Inhibition by clebopride and Delagrange 2674 of [3H]-FNM binding was apparently competitive and readily reversible. 4. In the presence of gamma-aminobutyric acid (GABA), the ability of diazepam and Delagrange 2674 to displace [3H]-Ro 15-1788 binding was increased 3.6 and 1.6 fold respectively, compared to the absence of GABA, while ethyl beta-carboline-3-carboxylate (beta CCE) and clebopride were less potent in the presence of GABA. 5. Diazepam was 30 fold less potent at displacing [3H]-Ro 15-1788 in membranes that had been photoaffinity labelled with FNM than in control membranes, whereas the potency of beta CCE did not differ. Clebopride and Delagrange 2674 showed a less than two fold loss of potency in photoaffinity labelled membranes. 6. The pattern of binding of clebopride and Delagrange 2674 in these in vitro tests is similar to that found previously with partial agonists or antagonists at BDZ binding sites. 7. Clebopride and Delagrange 2674 inhibited [3H]-FNM binding with similar potency in rat cerebellar and hippocampal membranes, suggesting they have no selectivity for BDZ1 and BDZ2 binding sites. 8. Clebopride and Delagrange 2674 are structurally dissimilar to other BDZ ligands and represent another chemical structure to probe brain BDZ binding sites.

Development of a Novel Tetravalent Synthetic Peptide That Binds to Phosphatidic Acid.

PubMed

Ogawa, Rina; Nagao, Kohjiro; Taniuchi, Kentaro; Tsuchiya, Masaki; Kato, Utako; Hara, Yuji; Inaba, Takehiko; Kobayashi, Toshihide; Sasaki, Yoshihiro; Akiyoshi, Kazunari; Watanabe-Takahashi, Miho; Nishikawa, Kiyotaka; Umeda, Masato

2015-01-01

We employed a multivalent peptide-library screening technique to identify a peptide motif that binds to phosphatidic acid (PA), but not to other phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). A tetravalent peptide with the sequence motif of MARWHRHHH, designated as PAB-TP (phosphatidic acid-binding tetravalent peptide), was shown to bind as low as 1 mol% of PA in the bilayer membrane composed of PC and cholesterol. Kinetic analysis of the interaction between PAB-TP and the membranes containing 10 mol% of PA showed that PAB-TP associated with PA with a low dissociation constant of KD = 38 ± 5 nM. Coexistence of cholesterol or PE with PA in the membrane enhanced the PAB-TP binding to PA by increasing the ionization of the phosphomonoester head group as well as by changing the microenvironment of PA molecules in the membrane. Amino acid replacement analysis demonstrated that the tryptophan residue at position 4 of PAB-TP was involved in the interaction with PA. Furthermore, a series of amino acid substitutions at positions 5 to 9 of PAB-TP revealed the involvement of consecutive histidine and arginine residues in recognition of the phosphomonoester head group of PA. Our results demonstrate that the recognition of PA by PAB-TP is achieved by a combination of hydrophobic, electrostatic and hydrogen-bond interactions, and that the tetravalent structure of PAB-TP contributes to the high affinity binding to PA in the membrane. The novel PA-binding tetravalent peptide PAB-TP will provide insight into the molecular mechanism underlying the recognition of PA by PA-binding proteins that are involved in various cellular events.

The Association of Myosin IB with Actin Waves in Dictyostelium Requires Both the Plasma Membrane-Binding Site and Actin-Binding Region in the Myosin Tail

PubMed Central

Brzeska, Hanna; Pridham, Kevin; Chery, Godefroy; Titus, Margaret A.; Korn, Edward D.

2014-01-01

F-actin structures and their distribution are important determinants of the dynamic shapes and functions of eukaryotic cells. Actin waves are F-actin formations that move along the ventral cell membrane driven by actin polymerization. Dictyostelium myosin IB is associated with actin waves but its role in the wave is unknown. Myosin IB is a monomeric, non-filamentous myosin with a globular head that binds to F-actin and has motor activity, and a non-helical tail comprising a basic region, a glycine-proline-glutamine-rich region and an SH3-domain. The basic region binds to acidic phospholipids in the plasma membrane through a short basic-hydrophobic site and the Gly-Pro-Gln region binds F-actin. In the current work we found that both the basic-hydrophobic site in the basic region and the Gly-Pro-Gln region of the tail are required for the association of myosin IB with actin waves. This is the first evidence that the Gly-Pro-Gln region is required for localization of myosin IB to a specific actin structure in situ. The head is not required for myosin IB association with actin waves but binding of the head to F-actin strengthens the association of myosin IB with waves and stabilizes waves. Neither the SH3-domain nor motor activity is required for association of myosin IB with actin waves. We conclude that myosin IB contributes to anchoring actin waves to the plasma membranes by binding of the basic-hydrophobic site to acidic phospholipids in the plasma membrane and binding of the Gly-Pro-Gln region to F-actin in the wave. PMID:24747353

Multiple Drug Transport Pathways through human P-Glycoprotein(†)

PubMed Central

McCormick, James W.; Vogel, Pia D.; Wise, John G.

2015-01-01

P-glycoprotein (P-gp) is a plasma membrane efflux pump that is commonly associated with therapy resistances in cancers and infectious diseases. P-gp can lower the intracellular concentrations of many drugs to subtherapeutic levels by translocating them out of the cell. Because of the broad range of substrates transported by P-gp, overexpression of P-gp causes multidrug resistance. We reported previously on dynamic transitions of P-gp as it moved through conformations based on crystal structures of homologous ABCB1 proteins using in silico targeted molecular dynamics techniques. We expanded these studies here by docking transport substrates to drug binding sites of P-gp in conformations open to the cytoplasm, followed by cycling the pump through conformations that opened to the extracellular space. We observed reproducible transport of two substrates, daunorubicin and verapamil, by an average of 11 to 12 Å through the plane of the membrane as P-gp progressed through a catalytic cycle. Methyl-pyrophosphate, a ligand that should not be transported by P-gp, did not show this movement through P-gp. Drug binding to either of two subsites on P-gp appeared to determine the initial pathway used for drug movement through the membrane. The specific side-chain interactions with drugs within each pathway seemed to be, at least in part, stochastic. The docking and transport properties of a P-gp inhibitor, tariquidar, were also studied. A mechanism of inhibition by tariquidar is presented that involves stabilization of an outward open conformation with tariquidar bound in intracellular loops or at the drug binding domain of P-gp. PMID:26125482

Multiple Drug Transport Pathways through Human P-Glycoprotein.

PubMed

McCormick, James W; Vogel, Pia D; Wise, John G

2015-07-21

P-Glycoprotein (P-gp) is a plasma membrane efflux pump that is commonly associated with therapy resistances in cancers and infectious diseases. P-gp can lower the intracellular concentrations of many drugs to subtherapeutic levels by translocating them out of the cell. Because of the broad range of substrates transported by P-gp, overexpression of P-gp causes multidrug resistance. We reported previously on dynamic transitions of P-gp as it moved through conformations based on crystal structures of homologous ABCB1 proteins using in silico targeted molecular dynamics techniques. We expanded these studies here by docking transport substrates to drug binding sites of P-gp in conformations open to the cytoplasm, followed by cycling the pump through conformations that opened to the extracellular space. We observed reproducible transport of two substrates, daunorubicin and verapamil, by an average of 11-12 Å through the plane of the membrane as P-gp progressed through a catalytic cycle. Methylpyrophosphate, a ligand that should not be transported by P-gp, did not show this movement through P-gp. Drug binding to either of two subsites on P-gp appeared to determine the initial pathway used for drug movement through the membrane. The specific side-chain interactions with drugs within each pathway seemed to be, at least in part, stochastic. The docking and transport properties of a P-gp inhibitor, tariquidar, were also studied. A mechanism of inhibition by tariquidar that involves stabilization of an outward open conformation with tariquidar bound in intracellular loops or at the drug binding domain of P-gp is presented.

Binding, uptake, and transport of hypericin by Caco-2 cell monolayers.

PubMed

Sattler, S; Schaefer, U; Schneider, W; Hoelzl, J; Lehr, C M

1997-10-01

The biological evaluation of hypericin in various test models is hampered by its very poor water solubility. In the present study cyclodextrin formulations and liposomal preparations were investigated for improved delivery and solubility of hypericin in aqueous buffer systems. Caco-2 cells, grown to tight monolayers on 96-well tissue culture plates as well as on Transwell polycarbonate filters, were used to study the membrane binding and the epithelial transport of hypericin. Cumulative transport of hypericin, which could not be measured without the use of cyclodextrins, in apical-to-basolateral direction from cyclodextrin-hypericin buffer solutions was 3-5% at 37 degrees C and approximately 0.12% at 4 degrees C after 5 h. After an incubation time of 1 h at 37 and 4 degrees C, 12.7% +/- 2.6% and 6.5% +/- 0.8%, respectively, of hypericin were found to be bound to or taken up by Caco-2 cells. Liposomal formulations markedly increased the solubility of hypericin in Krebs-Ringer buffer, but there was no effect observed on the binding and transport of hypericin delivered by liposomes in the Caco-2 cell model. Due to the fluorescence properties of hypericin, its interaction with the cells could be visualized by confocal laser scanning microscopy. The results indicate that a significant accumulation of the drug in the cell membrane and the cell nucleus membrane takes place. We conclude that hypericin is absorbed through the intestinal epithelium by passive transcellular diffusion and that increasing its solubility by cyclodextrin appears as a promising approach to increase its oral bioavailability for pharmaceutical formulations.

Comparison of kinetics, toxicity, oligomers formation and membrane binding capacity of α-synuclein familial mutations at A53 site including newly discovered A53V mutation.

PubMed

Mohite, Ganesh M; Kumar, Rakesh; Panigrahi, Rajlaxmi; Navalkar, Ambuja; Singh, Nitu; Datta, Debalina; Mehra, Surabhi; Ray, Soumik; Gadhe, Laxmikant G; Das, Subhadeep; Singh, Namrata; Chatterjee, Debdeep; Kumar, Ashutosh; Maji, Samir K

2018-05-17

The involvement of α-synuclein (α-Syn) amyloid formation in Parkinson's disease (PD) pathogenesis is supported by the discovery of α-Syn gene (SNCA) mutations linked with familial PD, which are known to modulate the oligomerization and aggregation of α-Syn. Recently, the A53V mutation has been discovered, which leads to the late-onset PD. In the present study, we characterized for the first time the biophysical properties including the aggregation propensities, toxicity of aggregated species and membrane binding capability of A53V along with all familial mutations at A53 position. Present data suggest that A53V accelerate fibrillation of α-Syn without affecting the overall morphology and cytotoxicity of fibrils compared to wild-type protein. The aggregation propensity for A53 mutants is found to be; A53T>A53V>WT>A53E. Further, time course aggregation study reveals that A53V mutant promotes early oligomerization similar to A53T mutation. It promotes the highest amount of oligomer formation immediate after dissolution, which are cytotoxic. Although in the presence of membrane-mimicking environments, A53V mutation showed similar extent of helix-induction capacity as of WT protein, however, it exhibited lesser binding to lipid vesicle. The NMR study revealed unique chemical shift perturbation by A53V mutation com-pared to other mutations at A53 site. The present study might help to establish the disease-causing mechanism of A53V in PD pathology.

Rotenone Activates Phagocyte NADPH Oxidase through Binding to Its Membrane Subunit gp91phox

PubMed Central

Zhou, Hui; Zhang, Feng; Chen, Shih-heng; Zhang, Dan; Wilson, Belinda; Hong, Jau-shyong; Gao, Hui-Ming

2011-01-01

Rotenone, a widely used pesticide, reproduces Parkinsonism in rodents and associates with increased risk for Parkinson’s disease. We previously reported rotenone increased superoxide production through stimulating microglial phagocyte NADPH oxidase (PHOX). The present study identified a novel mechanism by which rotenone activates PHOX. Ligand-binding assay revealed that rotenone directly bound to membrane gp91phox, the catalytic subunit of PHOX; such binding was inhibited by diphenyleneiodonium, a PHOX inhibitor with a binding site on gp91phox. Functional studies showed both membrane and cytosolic subunits were required for rotenone-induced superoxide production in cell-free systems, intact phagocytes, and COS7 cells transfected with membrane subunits (gp91phox/p22phox) and cytosolic subunits (p67phox and p47phox). Rotenone-elicited extracellular superoxide release in p47phox-deficient macrophages suggested rotenone enabled to activate PHOX through a p47phox-independent mechanism. Increased membrane translocation of p67phox, elevated binding of p67phox to rotenone-treated membrane fractions, and co-immunoprecipitation of p67phox and gp91phox in rotenone-treated wild-type and p47phox-deficient macrophages indicated p67phox played a critical role in rotenone-induced PHOX activation via its direct interaction with gp91phox. Rac1, a Rho-like small GTPase, enhanced p67phox-gp91phox interaction; Rac1 inhibition decreased rotenone-elicited superoxide release. In conclusion, rotenone directly interacted with gp91phox; such an interaction triggered membrane translocation of p67phox, leading to PHOX activation and superoxide production. PMID:22094225

Structure-Activity Relationships of Antimicrobial and Lipoteichoic Acid-Sequestering Properties in Polyamine Sulfonamides ▿

PubMed Central

Warshakoon, Hemamali J.; Burns, Mark R.; David, Sunil A.

2009-01-01

We have recently confirmed that lipoteichoic acid (LTA), a major constituent of the gram-positive bacterial surface, is the endotoxin of gram-positive bacteria that induces proinflammatory molecules in a Toll-like receptor 2 (TLR2)-dependent manner. LTA is an anionic amphipath whose physicochemical properties are similar to those of lipopolysaccharide (LPS), which is found on the outer leaflet of the outer membranes of gram-negative organisms. Hypothesizing that compounds that sequester LPS could also bind to and inhibit LTA-induced cellular activation, we screened congeneric series of polyamine sulfonamides which we had previously shown effectively neutralized LPS both in vitro and in animal models of endotoxemia. We observed that these compounds do bind to and neutralize LTA, as reflected by the inhibition of TLR2-mediated NF-κB induction in reporter gene assays. Structure-activity studies showed a clear dependence of the acyl chain length on activity against LTA in compounds with spermine and homospermine scaffolds. We then sought to examine possible correlations between the neutralizing potency toward LTA and antimicrobial activity in Staphylococcus aureus. A linear relationship between LTA sequestration activity and antimicrobial activity for compounds with a spermine backbone was observed, while all compounds with a homospermine backbone were equally active against S. aureus, regardless of their neutralizing potency toward LTA. These results suggest that the number of protonatable charges is a key determinant of the activity toward the membranes of gram-positive bacteria. The development of resistance to membrane-active antibiotics has been relatively slower than that to conventional antibiotics, and it is possible that compounds such as the acylpolyamines may be useful clinically, provided that they have an acceptable safety profile and margin of safety. A more detailed understanding of the mechanisms of interactions of these compounds with LPS and LTA, as well as the gram-negative and -positive bacterial cell surfaces, will be instructive and should allow the rational design of analogues which combine antisepsis and antibacterial properties. PMID:18955537

Measurement of Epidermal Growth Factor Receptor-Derived Signals Within Plasma Membrane Clathrin Structures.

PubMed

Lucarelli, Stefanie; Delos Santos, Ralph Christian; Antonescu, Costin N

2017-01-01

The epidermal growth factor (EGF) receptor (EGFR) is an important regulator of cell growth, proliferation, survival, migration, and metabolism. EGF binding to EGFR triggers the activation of the receptor's intrinsic kinase activity, in turn eliciting the recruitment of many secondary signaling proteins and activation of downstream signals, such as the activation of phosphatidylinositol-3-kinase (PI3K) and Akt, a process requiring the phosphorylation of Gab1. While the identity of many signals that can be activated by EGFR has been revealed, how the spatiotemporal organization of EGFR signaling within cells controls receptor outcome remains poorly understood. Upon EGF binding at the plasma membrane, EGFR is internalized by clathrin-mediated endocytosis following recruitment to clathrin-coated pits (CCPs). Further, plasma membrane CCPs, but not EGFR internalization, are required for EGF-stimulated Akt phosphorylation. Signaling intermediates such as phosphorylated Gab1, which lead to Akt phosphorylation, are enriched within CCPs upon EGF stimulation. These findings indicate that some plasma membrane CCPs also serve as signaling microdomains required for certain facets of EGFR signaling and are enriched in key EGFR signaling intermediates. Understanding how the spatiotemporal organization of EGFR signals within CCP microdomains controls receptor signaling outcome requires imaging methods that can systematically resolve and analyze the properties of CCPs, EGFR and key signaling intermediates. Here, we describe methods using total internal reflection fluorescence microscopy imaging and analysis to systematically study the enrichment of EGFR and key EGFR-derived signals within CCPs.

A Monomeric Membrane Peptide that Lives in Three Worlds: In Solution, Attached to, and Inserted across Lipid Bilayers

PubMed Central

Reshetnyak, Yana K.; Segala, Michael; Andreev, Oleg A.; Engelman, Donald M.

2007-01-01

The membrane peptide pH (low) insertion peptide (pHLIP) lives in three worlds, being soluble in aqueous solution at pH 7.4, binding to the surface of lipid bilayers, and inserting as a transbilayer helix at low pH. With low pH driving the process, pHLIP can translocate cargo molecules attached to its C-terminus via a disulfide and release them in the cytoplasm of a cell. Here we examine a key aspect of the mechanism, showing that pHLIP is monomeric in each of its three major states: soluble in water near neutral pH (state I), bound to the surface of a membrane near neutral pH (state II), and inserted across the membrane as an α-helix at low pH (state III). The peptide does not induce fusion or membrane leakage. The unique properties of pHLIP made it attractive for the biophysical investigation of membrane protein folding in vitro and for the development of a novel class of delivery peptides for the transport of therapeutic and diagnostic agents to acidic tissue sites associated with various pathological processes in vivo. PMID:17557792

Membrane transporters studied by EPR spectroscopy: structure determination and elucidation of functional dynamics.

PubMed

Mullen, Anna; Hall, Jenny; Diegel, Janika; Hassan, Isa; Fey, Adam; MacMillan, Fraser

2016-06-15

During their mechanistic cycles membrane transporters often undergo extensive conformational changes, sampling a range of orientations, in order to complete their function. Such membrane transporters present somewhat of a challenge to conventional structural studies; indeed, crystallization of membrane-associated proteins sometimes require conditions that vary vastly from their native environments. Moreover, this technique currently only allows for visualization of single selected conformations during any one experiment. EPR spectroscopy is a magnetic resonance technique that offers a unique opportunity to study structural, environmental and dynamic properties of such proteins in their native membrane environments, as well as readily sampling their substrate-binding-induced dynamic conformational changes especially through complementary computational analyses. Here we present a review of recent studies that utilize a variety of EPR techniques in order to investigate both the structure and dynamics of a range of membrane transporters and associated proteins, focusing on both primary (ABC-type transporters) and secondary active transporters which were key interest areas of the late Professor Stephen Baldwin to whom this review is dedicated. © 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Lipid-Mediated Targeting with Membrane Wrapped Nanoparticles in the Presence of Corona Formation

PubMed Central

Xu, Fangda; Reiser, Michael; Yu, Xinwei; Gummuluru, Suryaram; Wetzler, Lee; Reinhard, Björn M.

2016-01-01

Membrane wrapped nanoparticles represent a versatile platform for utilizing specific lipid-receptor interactions, such as siallyllactose-mediated binding of the ganglioside GM3 to Siglec1 (CD169), for targeting purposes. The membrane wrap around the nanoparticles does not only serve as a matrix to incorporate GM3 as targeting moiety for antigen presenting cells but also offers unique opportunities for constructing a biomimetic surface from lipids with potentially protein repellent properties. We characterize non-specific protein adsorption (corona formation) to membrane wrapped nanoparticles with core diameters of approx. 35 nm and 80 nm and its effect on the GM3-mediated targeting efficacy as function of surface charge through combined in vitro and in vivo studies. The stability and fate of the membrane wrap around the nanoparticles in a simulated biological fluid and after uptake in CD169 expressing antigen presenting cells is experimentally tested. Finally, we demonstrate in hock immunization studies in mice that GM3 decorated membrane wrapped nanoparticles achieve a selective enrichment in the peripheral regions of popliteal lymph nodes that contain high concentrations of CD169 expressing antigen presenting cells. PMID:26720275

Location and ion-binding of membrane-associated valinomycin, a proton nuclear magnetic resonance study.

PubMed

Meers, P; Feigenson, G W

1988-03-03

Valinomycin, incorporated in small unilamellar vesicles of perdeuterated dimyristoylphosphatidylcholine, reveals several well-resolved 1H-NMR resonances. These resonances were used to examine the location, orientation and ion-binding of membrane-bound valinomycin. The order of affinity of membrane-bound valinomycin for cations is Rb+ greater than K+ greater than Cs+ greater than Ba2+, and binding is sensitive to surface change. The exchange between bound and free forms is fast on the NMR time scale. The intrinsic binding constants, extrapolated to zero anion concentration, are similar to those determined in aqueous solution. Rb+ and K+ show 1:1 binding to valinomycin, whereas the stoichiometry of Cs+ and Ba2+ is not certain. Paramagnetic chemical shift reagents and nitroxide spin label relaxation probes were used to study the location and orientation of valinomycin in the membrane. Despite relatively fast exchange of bound cations, the time average location of the cation-free form of valinomycin is deep within the bilayer under the conditions of these experiments. Upon complexation to K+, valinomycin moves closer to the interfacial region.

A Structure-Based Mechanism for Arf1-Dependent Recruitment of Coatomer to Membranes

PubMed Central

Yu, Xinchao; Breitman, Marianna; Goldberg, Jonathan

2012-01-01

Summary Budding of COPI-coated vesicles from Golgi membranes requires an Arf-family G protein and the coatomer complex recruited from cytosol. Arf is also required with coatomer-related clathrin adaptor complexes to bud vesicles from the trans-Golgi network and endosomal compartments. To understand the structural basis for Arf-dependent recruitment of a vesicular coat to the membrane, we determined the structure of Arf1 bound to the γζ-COP subcomplex of coatomer. Structure-guided biochemical analysis reveals that a second Arf1-GTP molecule binds to βδ-COP at a site common to the γ- and β-COP subunits. The Arf1-binding sites on coatomer are spatially related to PtdIns4,5P2-binding sites on the endocytic AP2 complex, providing evidence that the orientation of membrane binding is general for this class of vesicular coat proteins. A bivalent GTP-dependent binding mode has implications for the dynamics of coatomer interaction with the Golgi and for the selection of cargo molecules. PMID:22304919

Interactions between antimicrobial polynorbornenes and phospholipid vesicles monitored by light scattering and microcalorimetry.

PubMed

Gabriel, Gregory J; Pool, Joanna G; Som, Abhigyan; Dabkowski, Jeffrey M; Coughlin, E Bryan; Muthukumar, M; Tew, Gregory N

2008-11-04

Antimicrobial polynorbornenes composed of facially amphiphilic monomers have been previously reported to accurately emulate the antimicrobial activity of natural host-defense peptides (HDPs). The lethal mechanism of most HDPs involves binding to the membrane surface of bacteria leading to compromised phospholipid bilayers. In this paper, the interactions between biomimetic vesicle membranes and these cationic antimicrobial polynorbornenes are reported. Vesicle dye-leakage experiments were consistent with previous biological assays and corroborated a mode of action involving membrane disruption. Dynamic light scattering (DLS) showed that these antimicrobial polymers cause extensive aggregation of vesicles without complete bilayer disintegration as observed with surfactants that efficiently solubilize the membrane. Fluorescence microscopy on vesicles and bacterial cells also showed polymer-induced aggregation of both synthetic vesicles and bacterial cells. Isothermal titration calorimetry (ITC) afforded free energy of binding values (Delta G) and polymer to lipid binding ratios, plus revealed that the interaction is entropically favorable (Delta S>0, Delta H>0). It was observed that the strength of vesicle binding was similar between the active polymers while the binding stoichiometries were dramatically different.

Expression of senescent antigen on erythrocytes infected with a knobby variant of the human malaria parasite Plasmodium falciparum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Winograd, E.; Greenan, J.R.T.; Sherman, I.W.

Erythrocytes infected with a knobby variant of Plasmodium falciparum selectively bind IgG autoantibodies in normal human serum. Quantification of membrane-bound IgG, by use of /sup 125/I-labeled protein A, revealed that erythrocytes infected with the knobby variant bound 30 times more protein A than did noninfected erythrocytes; infection with a knobless variant resulted in less than a 2-fold difference compared with noninfected erythrocytes. IgG binding to knobby erythrocytes appeared to be related to parasite development, since binding of /sup 125/I-labeled protein A to cells bearing young trophozoites (less than 20 hr after parasite invasion) was similar to binding to uninfected erythrocytes.more » By immunoelectron microscopy, the membrane-bound IgG on erythrocytes infected with the knobby variant was found to be preferentially associated with the protuberances (knobs) of the plasma membrane. The removal of aged or senescent erythrocytes from the peripheral circulation is reported to involve the binding of specific antibodies to an antigen (senescent antigen) related to the major erythrocyte membrane protein band 3. Since affinity-purified autoantibodies against band 3 specifically bound to the plasma membrane of erythrocytes infected with the knobby variant of P. falciparum, it is clear that the malaria parasite induces expression of senescent antigen.« less

Development of a μO-Conotoxin Analogue with Improved Lipid Membrane Interactions and Potency for the Analgesic Sodium Channel NaV1.8*

PubMed Central

Deuis, Jennifer R.; Dekan, Zoltan; Inserra, Marco C.; Lee, Tzong-Hsien; Aguilar, Marie-Isabel; Craik, David J.; Lewis, Richard J.; Alewood, Paul F.; Mobli, Mehdi; Schroeder, Christina I.; Henriques, Sónia Troeira; Vetter, Irina

2016-01-01

The μO-conotoxins MrVIA, MrVIB, and MfVIA inhibit the voltage-gated sodium channel NaV1.8, a well described target for the treatment of pain; however, little is known about the residues or structural elements that define this activity. In this study, we determined the three-dimensional structure of MfVIA, examined its membrane binding properties, performed alanine-scanning mutagenesis, and identified residues important for its activity at human NaV1.8. A second round of mutations resulted in (E5K,E8K)MfVIA, a double mutant with greater positive surface charge and greater affinity for lipid membranes compared with MfVIA. This analogue had increased potency at NaV1.8 and was analgesic in the mouse formalin assay. PMID:27026701

Continuous planar phospholipid bilayer supported on porous silicon thin film reflector.

PubMed

Cunin, Frédérique; Milhiet, Pierre-Emmanuel; Anglin, Emily; Sailor, Michael J; Espenel, Cédric; Le Grimellec, Christian; Brunel, Daniel; Devoisselle, Jean-Marie

2007-10-01

Reconstituting artificial membranes for in vitro studies of cell barrier mechanisms and properties is of major interest in biology. Here, artificial membranes supported on porous silicon photonic crystal reflectors are prepared and investigated. The materials are of interest for label-free probing of supported membrane events such as protein binding, molecular recognition, and transport. The porous silicon substrates are prepared as multilayered films consisting of a periodically varying porosity, with pore dimensions of a few nanometers in size. Planar phospholipid bilayers are deposited on the topmost surface of the oxidized hydrophilic mesoporous silicon films. Atomic force microscopy provides evidence of continuous bilayer deposition at the surface, and optical measurements indicate that the lipids do not significantly infiltrate the porous region. The presence of the supported bilayer does not obstruct the optical spectrum from the porous silicon layer, suggesting that the composite structures can act as effective optical biosensors.

Solvent relaxation of fluorescent labels as a new tool for the detection of polarity and rigidity changes in membranes

NASA Astrophysics Data System (ADS)

Hof, Martin; Hutterer, Rudi

1998-04-01

Since solvent relaxation (SR) exclusively depends on the physical properties of the dye environment, SR spectroscopy of defined located labels in amphiphilic assemblies accomplishes the characterisation of specific domains. The most accurate way to characterise SR is the determination of the time-dependent Stokes shift. The time course of the Stokes shift, expressed as a solvent relaxation time, gives information about both the rigidity and polarity of the dye environment. The absolute value of the Stokes shift following the excitation is correlated with the polarity of the probed region. The validity of this approach for the investigation of phospholipid bilayers is illustrated by listing the parameters influencing the SR kinetics of appropriate membrane labels: membrane curvature, percentage of phosphatidylserine (PS) in small unilamell vesicles (SUV), addition of Ca2+ ions, binding of vitamin-K dependent proteins, percentage of diether-lipids in phosphatidylcholine (PC)-vesicles, and temperature.

Lipid-binding analysis using a fat blot assay.

PubMed

Munnik, Teun; Wierzchowiecka, Magdalena

2013-01-01

Protein-lipid interactions play an important role in lipid metabolism, membrane trafficking and cell -signaling by regulating protein localization, activation, and function. The Fat Blot assay is a relatively simple and inexpensive method to examine these interactions using nitrocellulose membrane-immobilized lipids. The assay is adapted from the method by Dowler et al. (Sci STKE 129:pl6, 2002) and provides qualitative and quantitative information on the relative affinity with which a protein binds to a particular lipid. To perform a Fat Blot assay, serial dilutions of different phospholipids are spotted onto a nitrocellulose membrane. These membranes are then incubated with a lipid-binding protein possessing a GST (or other epitope) tag. The membranes are washed and the protein, which is bound to the membrane by virtue of its interaction with the lipid's head group, is detected by immunoblotting with an antibody against GST (or other epitope). The procedure only requires a few micrograms of protein and is quick, simple and cheap to perform.

Relevant interactions of antimicrobial iron chelators and membrane models revealed by nuclear magnetic resonance and molecular dynamics simulations.

PubMed

Coimbra, João T S; Moniz, Tânia; Brás, Natércia F; Ivanova, Galya; Fernandes, Pedro A; Ramos, Maria J; Rangel, Maria

2014-12-18

The dynamics and interaction of 3-hydroxy-4-pyridinone fluorescent iron chelators, exhibiting antimicrobial properties, with biological membranes were evaluated through NMR and molecular dynamics simulations. Both NMR and MD simulation results support a strong interaction of the chelators with the lipid bilayers that seems to be strengthened for the rhodamine containing compounds, in particular for compounds that include ethyl groups and a thiourea link. For the latter type of compounds the interaction reaches the hydrophobic core of the lipid bilayer. The molecular docking and MD simulations performed for the potential interaction of the chelators with DC-SIGN receptors provide valuable information regarding the cellular uptake of these compounds since the results show that the fluorophore fragment of the molecular framework is essential for an efficient binding. Putting together our previous and present results, we put forward the hypothesis that all the studied fluorescent chelators have access to the cell, their uptake occurs through different pathways and their permeation properties correlate with a better access to the cell and its compartments and, consequently, with the chelators antimicrobial properties.

Structural and mechanistic insights into phospholipid transfer by Ups1–Mdm35 in mitochondria

PubMed Central

Watanabe, Yasunori; Tamura, Yasushi; Kawano, Shin; Endo, Toshiya

2015-01-01

Eukaryotic cells are compartmentalized into membrane-bounded organelles whose functions rely on lipid trafficking to achieve membrane-specific compositions of lipids. Here we focused on the Ups1–Mdm35 system, which mediates phosphatidic acid (PA) transfer between the outer and inner mitochondrial membranes, and determined the X-ray structures of Mdm35 and Ups1–Mdm35 with and without PA. The Ups1–Mdm35 complex constitutes a single domain that has a deep pocket and flexible Ω-loop lid. Structure-based mutational analyses revealed that a basic residue at the pocket bottom and the Ω-loop lid are important for PA extraction from the membrane following Ups1 binding. Ups1 binding to the membrane is enhanced by the dissociation of Mdm35. We also show that basic residues around the pocket entrance are important for Ups1 binding to the membrane and PA extraction. These results provide a structural basis for understanding the mechanism of PA transfer between mitochondrial membranes. PMID:26235513

Testing the limits of rational design by engineering pH sensitivity into membrane-active peptides.

PubMed

Wiedman, Gregory; Wimley, William C; Hristova, Kalina

2015-04-01

In this work, we sought to rationally design membrane-active peptides that are triggered by low pH to form macromolecular-sized pores in lipid bilayers. Such peptides could have broad utility in biotechnology and in nanomedicine as cancer therapeutics or drug delivery vehicles that promote release of macromolecules from endosomes. Our approach to rational design was to combine the properties of a pH-independent peptide, MelP5, which forms large pores allowing passage of macromolecules, with the properties of two pH-dependent membrane-active peptides, pHlip and GALA. We created two hybrid sequences, MelP5_Δ4 and MelP5_Δ6, by using the distribution of acidic residues on pHlip and GALA as a guide to insert acidic amino acids into the amphipathic helix of MelP5. We show that the new peptides bind to lipid bilayers and acquire secondary structure in a pH-dependent manner. The peptides also destabilize bilayers in a pH-dependent manner, such that lipid vesicles release the small molecules ANTS/DPX at low pH only. Thus, we were successful in designing pH-triggered pore-forming peptides. However, no macromolecular release was observed under any conditions. Therefore, we abolished the unique macromolecular poration properties of MelP5 by introducing pH sensitivity into its sequence. We conclude that the properties of pHlip, GALA, and MelP5 are additive, but only partially so. We propose that this lack of additivity is a limitation in the rational design of novel membrane-active peptides, and that high-throughput approaches to discovery will be critical for continued progress in the field. Copyright © 2015 Elsevier B.V. All rights reserved.

The sodium channel in membranes of electroplax. Binding of batrachotoxinin-a [(3)H]benzoate to particulate preparations from electric eel (electrophorus).

PubMed

McNeal, E T; Daly, J W

1986-01-01

Batrachotoxinin-A [(3)H]benzoate ([(3)H]BTX-B) binds specifically and with high affinity (K(D) 48 nM) to sites (B(max) 2.1 pmol/mg protein) associated with voltage-dependent sodium channels in rodent brain vesicular preparations. High affinity binding requires the presence of scorpion (Leiurus) venom and a membrane potential. Local anesthetics antagonize the binding. Nonspecific binding is defined in the presence of veratridine. In particulate preparations from electroplax of the eel Electrophorus electricus, [(3)H]BTX-B binds with a K(D) of about 140 nM and a B(max) of 2.5 pmol/mg protein in the presence of scorpion venom. Higher concentrations of scorpion venom are required to enhance binding in Electrophorus preparations than in brain preparations. Local anesthetics antagonize binding in Electrophorus preparations with potencies similar to those in brain preparations. Veratridine and batrachotoxin are less potent in blocking binding in Electrophorus than in brain preparations. It appears likely that binding in Electrophorus preparations is primarily to membrane fragments rather than vesicular entities as in brain. Binding of [(3)H]BTX-B to particulate preparations from electroplax of the ray Torpedo californica and the catfish Malapterurus electricus is mainly nonspecific. Scorpion venom does not enhance total binding and local anesthetics are not effective in antagonizing binding.

Aerobic sn-glycerol-3-phosphate dehydrogenase from Escherichia coli binds to the cytoplasmic membrane through an amphipathic alpha-helix.

PubMed Central

Walz, Antje-Christine; Demel, Rudy A; de Kruijff, Ben; Mutzel, Rupert

2002-01-01

sn-Glycerol-3-phosphate dehydrogenase (GlpD) from Escherichia coli is a peripheral membrane enzyme involved in respiratory electron transfer. For it to display its enzymic activity, binding to the inner membrane is required. The way the enzyme interacts with the membrane and how this controls activity has not been elucidated. In the present study we provide evidence for direct protein-lipid interaction. Using the monolayer technique, we observed insertion of GlpD into lipid monolayers with a clear preference for anionic phospholipids. GlpD variants with point mutations in their predicted amphipathic helices showed a decreased ability to penetrate anionic phospholipid monolayers. From these data we propose that membrane binding of GlpD occurs by insertion of an amphipathic helix into the acyl-chain region of lipids mediated by negatively charged phospholipids. PMID:11955283

Vitamin E: A Role in Signal Transduction.

PubMed

Zingg, Jean-Marc

2015-01-01

Vitamin E modulates the activity of several signal transduction enzymes with consequent alterations of gene expression. At the molecular level, vitamin E may directly bind to these enzymes and compete with their substrates, or it may change their activity by redox regulation. The translocation of several of these enzymes to the plasma membrane is regulated by vitamin E, suggesting the modulation of protein-membrane interactions as a common mechanism for vitamin E action. Enzyme-membrane interactions can be affected by vitamin E by interference with binding to specific membrane lipids or by altering cellular structures such as membrane microdomains (lipid rafts). Moreover, competition by vitamin E for common binding sites within lipid transport proteins may alter the traffic of lipid mediators and thus affect their signaling and enzymatic conversion. In this review, the main effects of vitamin E on enzymes involved in signal transduction are summarized and possible molecular mechanisms leading to enzyme modulation are evaluated.

Nanostructured Block Polymer Membranes as High Capacity Adsorbers for the Capture of Metal Ions from Water

NASA Astrophysics Data System (ADS)

Boudouris, Bryan; Weidman, Jacob; Mulvenna, Ryan; Phillip, William

The efficient removal of metal ions from aqueous streams is of significant import in applications ranging from industrial waste treatment to the purification of drinking water. An emerging paradigm associated with this separation is one that utilizes membrane adsorbers as a means by which to bind metal salt contaminants. Here, we demonstrate that the casting of an A-B-C triblock polymer using the self-assembly and non-solvent induced phase separation (SNIPS) methodology results in a nanoporous membrane geometry. The nature of the triblock polymer affords an extremely high density of binding sites within the membrane. As such, we demonstrate that the membranes with binding capacities equal to that of state-of-the-art packed bed columns. Moreover, because the affinity of the C moiety can be tuned, highly selective binding events can occur based solely on the chemistry of the block polymer and the metal ions in solution (i.e., in a manner that is independent of the size of the metal ions). Due to these combined facts, these membranes efficiently remove heavy metal (e.g., lead- and cadmium-based) salts from contaminated water streams with greater than 95% efficiency. Finally, we show that the membranes can be regenerated through a simple treatment in order to provide long-lasting adsorber systems as well. Thus, it is anticipated that these nanostructured triblock polymer membranes are a platform by which to obtain next-generation water purification processes.

Interaction of the Full-length Bax Protein with Biomimetic Mitochondrial Liposomes: A Small-Angle Neutron Scattering and Fluorescence Study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Satsoura, D; Kucerka, Norbert; Shivakumar, S

2012-01-01

In response to apoptotic stimuli, the pro-apoptotic protein Bax inserts in the outer mitochondrial membrane, resulting in the formation of pores and the release of several mitochondrial components, and sealing the cell's fate. To study the binding of Bax to membranes, we used an in vitro system consisting of 50 nm diameter liposomes prepared with a lipid composition mimicking that of mitochondrial membranes in which recombinant purified full-length Bax was inserted via activation with purified tBid. We detected the association of the protein with the membrane using fluorescence fluctuation methods, and found that it could well be described by anmore » equilibrium between soluble and membrane-bound Bax and that at a high protein-toliposome ratio the binding seemed to saturate at about 15 Bax proteins per 50 nm diameter liposome. We then obtained structural data for samples in this saturated binding regime using small-angle neutron scattering under different contrast matching conditions. Utilizing a simple model to fit the neutron data, we observed that a significant amount of the protein mass protrudes above the membrane, in contrast to the conjecture that all of the membrane-associated Bax states are umbrella-like. Upon protein binding, we also observed a thinning of the lipid bilayer accompanied by an increase in liposome radius, an effect reminiscent of the action of antimicrobial peptides on membranes.« less

Transcriptional Regulation, Metal Binding Properties and Structure of Pden1597, an Unusual Zinc Transport Protein from Paracoccus denitrificans*

PubMed Central

Handali, Melody; Neupane, Durga P.; Roychowdhury, Hridindu; Yukl, Erik T.

2015-01-01

ATP-binding cassette (ABC) transporters of the cluster 9 family are ubiquitous among bacteria and essential for acquiring Zn2+ and Mn2+ from the environment or, in the case of pathogens, from the host. These rely on a substrate-binding protein (SBP) to coordinate the relevant metal with high affinity and specificity and subsequently release it to a membrane permease for translocation into the cytoplasm. Although a number of cluster 9 SBP structures have been determined, the structural attributes conferring Zn2+ or Mn2+ specificity remain ambiguous. Here we describe the gene expression profile, in vitro metal binding properties, and crystal structure of a new cluster 9 SBP from Paracoccus denitrificans we have called AztC. Although all of our results strongly indicate Zn2+ over Mn2+ specificity, the Zn2+ ion is coordinated by a conserved Asp residue only observed to date as a metal ligand in Mn2+-specific SBPs. The unusual sequence properties of this protein are shared among close homologues, including members from the human pathogens Klebsiella pneumonia and Enterobacter aerogenes, and would seem to suggest a subclass of Zn2+-specific transporters among the cluster 9 family. In any case, the unusual coordination environment of AztC expands the already considerable range of those available to Zn2+-specific SBPs and highlights the presence of a His-rich loop as the most reliable indicator of Zn2+ specificity. PMID:25787075

Transcriptional regulation, metal binding properties and structure of Pden1597, an unusual zinc transport protein from Paracoccus denitrificans

DOE PAGES

Handali, Melody; Neupane, Durga P.; Roychowdhury, Hridindu; ...

2015-03-18

Here, ATP-binding cassette (ABC) transporters of the cluster 9 family are ubiquitous among bacteria and essential for acquiring Zn 2+ and Mn 2+ from the environment or, in the case of pathogens, from the host. These rely on a substrate-binding protein (SBP) to coordinate the relevant metal with high affinity and specificity and subsequently release it to a membrane permease for translocation into the cytoplasm. Although a number of cluster 9 SBP structures have been determined, the structural attributes conferring Zn 2+ or Mn 2+ specificity remain ambiguous. Here we describe the gene expression profile, in vitro metal binding properties,more » and crystal structure of a new cluster 9 SBP from Paracoccus denitrificans we have called AztC. Although all of our results strongly indicate Zn 2+ over Mn 2+ specificity, the Zn 2+ ion is coordinated by a conserved Asp residue only observed to date as a metal ligand in Mn 2+-specific SBPs. The unusual sequence properties of this protein are shared among close homologues, including members from the human pathogens Klebsiella pneumonia and Enterobacter aerogenes, and would seem to suggest a subclass of Zn 2+-specific transporters among the cluster 9 family. In any case, the unusual coordination environment of AztC expands the already considerable range of those available to Zn 2+-specific SBPs and highlights the presence of a His-rich loop as the most reliable indicator of Zn 2+ specificity.« less

Role of the Fast Kinetics of Pyroglutamate-Modified Amyloid-β Oligomers in Membrane Binding and Membrane Permeability

PubMed Central

2015-01-01

Membrane permeability to ions and small molecules is believed to be a critical step in the pathology of Alzheimer’s disease (AD). Interactions of oligomers formed by amyloid-β (Aβ) peptides with the plasma cell membrane are believed to play a fundamental role in the processes leading to membrane permeability. Among the family of Aβs, pyroglutamate (pE)-modified Aβ peptides constitute the most abundant oligomeric species in the brains of AD patients. Although membrane permeability mechanisms have been studied for full-length Aβ1–40/42 peptides, these have not been sufficiently characterized for the more abundant AβpE3–42 fragment. Here we have compared the adsorbed and membrane-inserted oligomeric species of AβpE3–42 and Aβ1–42 peptides. We find lower concentrations and larger dimensions for both species of membrane-associated AβpE3–42 oligomers. The larger dimensions are attributed to the faster self-assembly kinetics of AβpE3–42, and the lower concentrations are attributed to weaker interactions with zwitterionic lipid headgroups. While adsorbed oligomers produced little or no significant membrane structural damage, increased membrane permeabilization to ionic species is understood in terms of enlarged membrane-inserted oligomers. Membrane-inserted AβpE3–42 oligomers were also found to modify the mechanical properties of the membrane. Taken together, our results suggest that membrane-inserted oligomers are the primary species responsible for membrane permeability. PMID:24950761

Inhibition of (/sup 3/H)nitrendipine binding by phospholipase A/sub 2/

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goldman, M.E.; Pisano, J.J.

Phospholipase A/sub 2/ from several sources inhibited (/sup 3/H)nitrendipine binding to membranes from brain, heart and ileal longitudinal muscle. The enzymes from bee venom and Russell's viper venom were most potent, having IC/sub 50/ values of approximately 5 and 14 ng/ml, respectively, in all three membrane preparations. Inhibition of binding by bee venom phospholipase A/sub 2/ was time- and dose-dependent. Mastoparan, a known facilitator of phospholipase A/sub 2/ enzymatic activity, shifted the bee venom phospholipase A/sub 2/ dose-response curve to the left. Pretreatment of brain membranes with bee venom phospholipase A/sub 2/ (10 ng/ml) for 15 min caused a 2-foldmore » increase in the K/sub d/ without changing the B/sub max/ compared with untreated membranes. Extension of the preincubation period to 30 min caused no further increase in the K/sub d/ but significantly decreased the B/sub max/ to 71% the value for untreated membranes. (/sup 3/H)Nitrendipine, preincubated with bee venom phospholipase A/sub 2/, was recovered and found to be fully active, indicating that the phospholipase A/sub 2/ did not modify the ligand. It is concluded that phospholipase A/sub 2/ acts on the membrane at or near the (/sup 3/H)nitrendipine binding site and that phospholipids play a key role in the interactions of 1,4 dihydropyridine calcium channel antagonists with the dihydropyridine binding site. 33 references, 3 figures, 1 table.« less

Polyreactive Antibodies Plus Complement Enhance the Phagocytosis of Cells Made Apoptotic by UV-Light or HIV

PubMed Central

Zhou, Zhao-hua; Wild, Teresa; Xiong, Ying; Sylvers, Peter; Zhang, Yahong; Zhang, Luxia; Wahl, Larry; Wahl, Sharon M.; Kozlowski, Steven; Notkins, Abner L.

2013-01-01

Polyreactive antibodies are a major component of the natural antibody repertoire and are capable of binding a variety of structurally unrelated antigens. Many of the properties attributed to natural antibodies, in fact, are turning out to be due to polyreactive antibodies. In humans, each day, billions of cells undergo apoptosis. In the present experiments, we show by ImageStream technology that although polyreactive antibodies do not bind to live T cells they bind to both the plasma membrane and cytoplasm of late apoptotic cells, fix complement, generate the anaphylatoxin C5a and increase by as much as 5 fold complement-mediated phagocytosis by macrophages. Of particular importance, T cells undergoing apoptosis following infection with HIV also bind polyreactive antibodies and are phagocytosed. We conclude that the polyreactive antibodies in the natural antibody repertoire contribute in a major way to the clearance of cells made apoptotic by a variety of natural and infectious processes. PMID:23881356

Interaction of KMP-11 with Phospholipid Membranes and Its Implications in Leishmaniasis: Effects of Single Tryptophan Mutations and Cholesterol.

PubMed

Sannigrahi, Achinta; Maity, Pabitra; Karmakar, Sanat; Chattopadhyay, Krishnananda

2017-03-02

KMP-11 is a small protein that is believed to control the overall bilayer pressure of the Leishmania parasite. Recent results have suggested that membrane binding and the presence of cholesterol affect the efficacy of Leishmanial infection, in which KMP-11 plays an important role. Nevertheless, there exists no systematic study of membrane interaction with KMP-11 either in the absence or presence of cholesterol. In this article, we investigated the interaction between KMP-11 and phospholipid membranes using an unsaturated (PC 18:1; 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)) and saturated (PC 12:0; 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC)) lipid as membrane mimics. Additionally, we studied the effect of cholesterol on the protein-membrane interaction. Steady-state as well as time-resolved fluorescence spectroscopy, isothermal titration calorimetry (ITC), and ζ-potential measurements were used for the determination of the binding constants for the wild-type (WT) and single-site tryptophan mutants. Single-site tryptophan mutants were designed to make sure that the tryptophan residues sample different surface exposures in different mutants. In the absence of cholesterol, the membrane-binding affinities of the partially exposed and buried tryptophan mutants (Y5W and Y48W, respectively) were found to be greater than those of the WT protein. In the presence of cholesterol, the binding constants of the WT and Y48W mutant were found to decrease with an increase in cholesterol concentration. This was in contrast to that in the Y5W and F77W mutants, in which the binding constants increased on adding cholesterol. The present study highlights the interplay among the conformational architecture of a protein, its interaction with the membrane, and membrane composition in modulating the survival of a Leishmania parasite inside host macrophages.

Characterization and antifungal properties of wheat nonspecific lipid transfer proteins.

PubMed

Sun, Jin-Yue; Gaudet, Denis A; Lu, Zhen-Xiang; Frick, Michele; Puchalski, Byron; Laroche, André

2008-03-01

This study simultaneously considered the phylogeny, fatty acid binding ability, and fungal toxicity of a large number of monocot nonspecific lipid transfer proteins (ns-LTP). Nine novel full-length wheat ns-LTP1 clones, all possessing coding sequences of 348 bp, isolated from abiotic- and biotic-stressed cDNA libraries from aerial tissues, exhibited highly conserved coding regions with 78 to 99 and 71 to 100% identity at the nucleotide and amino acid levels, respectively. Phylogenetic analyses revealed two major ns-LTP families in wheat. Eight wheat ns-LTP genes from different clades were cloned into the expression vector pPICZalpha and transformed into Pichia pastoris. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blotting, and in vitro lipid binding activity assay confirmed that the eight ns-LTP were all successfully expressed and capable of in vitro binding fatty acid molecules. A comparative in vitro study on the toxicity of eight wheat ns-LTP to mycelium growth or spore germination of eight wheat pathogens and three nonwheat pathogens revealed differential toxicities among different ns-LTP. Values indicating 50% inhibition of fungal growth or spore germination of three selected ns-LTP against six fungi ranged from 1 to 7 microM. In vitro lipid-binding activity of ns-LTP was not correlated with their antifungal activity. Using the fluorescent probe SYTOX Green as an indicator of fungal membrane integrity, the in vitro toxicity of wheat ns-LTP was associated with alteration in permeability of fungal membranes.

Epstein-Barr virus/complement fragment C3d receptor (CR2) reacts with p53, a cellular antioncogene-encoded membrane phosphoprotein: detection by polyclonal anti-idiotypic anti-CR2 antibodies.

PubMed Central

Barel, M; Fiandino, A; Lyamani, F; Frade, R

1989-01-01

Epstein-Barr virus and the C3d fragment of the third component of complement are specific extracellular ligands for complement receptor type 2 (CR2). However, intracellular proteins that react specifically with CR2 and are involved in post-membrane signals remain unknown. We recently prepared polyclonal anti-idiotypic anti-CR2 antibodies (Ab2) by using the highly purified CR2 molecule as original immunogen. We showed that Ab2 contained anti-idiotypic specificities that mimicked extracellular domains of CR2 and detected two distinct binding sites on CR2 for its specific extracellular ligands, Epstein-Barr virus and C3d. We postulated that Ab2 might also contain specificities that could mimic intracellular domains of CR2. Here we report that Ab2, which did not react with Raji B-lymphoma cell surface components, detected specifically, among all components solubilized from Raji cell membranes, a single intracellular membrane protein of apparent molecular mass of 53 kDa. This protein was identified as the p53 cellular antioncogene-encoded membrane phosphoprotein by analyzing its antigenic properties with Pab1801, a monoclonal anti-p53 antibody, and by comparing its biochemical properties with those of p53. Additionally, solubilized and purified CR2 bound to solubilized p53 immobilized on Pab1801-Sepharose. p53, like CR2, was localized only in purified plasma membranes and nuclei of Raji cells. These data suggest strongly that p53, a cellular antioncogene-encoded phosphoprotein, reacted specifically with CR2 in Raji membranes. This interaction may represent one of the important steps through which CR2 could be involved in human B-lymphocyte proliferation and transformation. Images PMID:2557614

The Antibiotic Novobiocin Binds and Activates the ATPase That Powers Lipopolysaccharide Transport.

PubMed

May, Janine M; Owens, Tristan W; Mandler, Michael D; Simpson, Brent W; Lazarus, Michael B; Sherman, David J; Davis, Rebecca M; Okuda, Suguru; Massefski, Walter; Ruiz, Natividad; Kahne, Daniel

2017-12-06

Novobiocin is an orally active antibiotic that inhibits DNA gyrase by binding the ATP-binding site in the ATPase subunit. Although effective against Gram-positive pathogens, novobiocin has limited activity against Gram-negative organisms due to the presence of the lipopolysaccharide-containing outer membrane, which acts as a permeability barrier. Using a novobiocin-sensitive Escherichia coli strain with a leaky outer membrane, we identified a mutant with increased resistance to novobiocin. Unexpectedly, the mutation that increases novobiocin resistance was not found to alter gyrase, but the ATPase that powers lipopolysaccharide (LPS) transport. Co-crystal structures, biochemical, and genetic evidence show novobiocin directly binds this ATPase. Novobiocin does not bind the ATP binding site but rather the interface between the ATPase subunits and the transmembrane subunits of the LPS transporter. This interaction increases the activity of the LPS transporter, which in turn alters the permeability of the outer membrane. We propose that novobiocin will be a useful tool for understanding how ATP hydrolysis is coupled to LPS transport.

Two widely expressed plasma membrane H(+)-ATPase isoforms of Nicotiana tabacum are differentially regulated by phosphorylation of their penultimate threonine.

PubMed

Bobik, Krzysztof; Duby, Geoffrey; Nizet, Yannick; Vandermeeren, Caroline; Stiernet, Patrick; Kanczewska, Justyna; Boutry, Marc

2010-04-01

The plasma membrane H(+)-ATPases PMA2 and PMA4 are the most widely expressed in Nicotiana plumbaginifolia, and belong to two different subfamilies. Both are activated by phosphorylation of a Thr at the penultimate position and the subsequent binding of 14-3-3 proteins. Their expression in Saccharomyces cerevisiae revealed functional and regulatory differences. To determine whether different regulatory properties between PMA2 and PMA4 exist in plants, we generated two monoclonal antibodies able to detect phosphorylation of the penultimate Thr of either PMA2 or PMA4 in a total protein extract. We also raised Nicotiana tabacum transgenic plants expressing 6-His-tagged PMA2 or PMA4, enabling their individual purification. Using these tools we showed that phosphorylation of the penultimate Thr of both PMAs was high during the early exponential growth phase of an N. tabacum cell culture, and then progressively declined. This decline correlated with decreased 14-3-3 binding and decreased plasma membrane ATPase activity. However, the rate and extent of the decrease differed between the two isoforms. Cold stress of culture cells or leaf tissues reduced the Thr phosphorylation of PMA2, whereas no significant changes in Thr phosphorylation of PMA4 were seen. These results strongly suggest that PMA2 and PMA4 are differentially regulated by phosphorylation. Analysis of the H(+)-ATPase phosphorylation status in leaf tissues indicated that no more than 44% (PMA2) or 32% (PMA4) was in the activated state under normal growth conditions. Purification of either isoform showed that, when activated, the two isoforms did not form hetero-oligomers, which is further support for these two H(+)-ATPase subfamilies having different properties.

Increased lectin binding capacity of trophoblastic cells of late day 5 rat blastocysts.

PubMed Central

Stein, B A; Shaw, T J; Turner, V F; Murphy, C R

1994-01-01

The binding of lectins to the trophoblast of rat blastocysts has been studied using quantitative ultrastructural cytochemistry. Rat blastocysts from early, mid and late d 5 of gestation were stained using biotinylated lectins (Phytolacca americana [Phy am], fucose binding protein [FBP] and soybean agglutinin [SBA]) and a sensitive avidin-ferritin cytochemical method. Electron micrographs of ferritin particles along the membrane were processed to produce images for which grey scale levels could be established and the ferritin particles automatically counted. The ferritin:membrane ratio was then calculated. Increased binding with Phy am (which detects short chain oligosaccharides) was found after midday of d 5, i.e. after hatching. Binding of FBP and SBA did not alter during the period studied. The increased concentration of oligosaccharides on the blastocyst surface membrane after hatching may have important implications for blastocyst attachment to the endometrium. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7649802

Topographical localization of the C-terminal region of the voltage-dependent sodium channel from Electrophorus electricus using antibodies raised against a synthetic peptide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gordon, R.D.; Fieles, W.E.; Schotland, D.L.

1987-01-01

A peptide corresponding to amino acid residues 1783-1794 near the C terminus of the electric eel sodium channel primary sequence of the eel (Electrophorus electricus) sodium channel has been synthesized and used to raise an antiserum in rabbits. This antiserum specifically recognized the peptide in a solid-phase radioimmunoassay. Specificity of the antiserum for the native channel protein was shown by its specific binding to a 280-kDa protein in immunoblots of eel electroplax membrane proteins. The antiserum also specifically labeled the innervated membrane of the eel electroplax in immunofluorescent studies. The membrane topology of the peptide recognized by this antiserum wasmore » proved in binding studies using oriented electroplax membrane vesicles. These vesicles were 98% right-side-out as determined by (/sup 3/H)saxitoxin binding. Binding of the antipeptide antiserum to this fraction was measured before and after permeabilization with 0.01% saponin. Specific binding to intact vesicles was low, but this binding increased 10-fold after permeabilization, implying a cytoplasmic orientation for the peptide. Confirmation for this orientation was then sought by localizing the antibody bound to intact electroplax cells with immunogold electron microscopy. The data imply that the region of the sodium channel primary sequence near the C terminus that is recognized by the anitserum is localized on the cytoplasmic side of the membrane; this localization provides some further constraints on models of sodium channel tertiary structure.« less

Binding of Sudan II and IV to lecithin liposomes and E. coli membranes: insights into the toxicity of hydrophobic azo dyes

PubMed Central

Li, Lu; Gao, Hong-Wen; Ren, Jiao-Rong; Chen, Ling; Li, Yu-Cheng; Zhao, Jian-Fu; Zhao, He-Ping; Yuan, Yuan

2007-01-01

Background Sudan red compounds are hydrophobic azo dyes, still used as food additives in some countries. However, they have been shown to be unsafe, causing tumors in the liver and urinary bladder in rats. They have been classified as category 3 human carcinogens by the International Agency for Research on Cancer. A number of hypotheses that could explain the mechanism of carcinogenesis have been proposed for dyes similar to the Sudan red compounds. Traditionally, investigations of the membrane toxicity of organic substances have focused on hydrocarbons, e.g. polycyclic aromatic hydrocarbons (PAHs), and DDT. In contrast to hydrocarbons, Sudan red compounds contain azo and hydroxy groups, which can form hydrogen bonds with the polar head groups of membrane phospholipids. Thus, entry may be impeded. They could have different toxicities from other lipophilic hydrocarbons. The available data show that because these compounds are lipophilic, interactions with hydrophobic parts of the cell are important for their toxicity. Lipophilic compounds accumulate in the membrane, causing expansion of the membrane surface area, inhibition of primary ion pumps and increased proton permeability. Results This work investigated the interactions of the amphiphilic compounds Sudan II and IV with lecithin liposomes and live Escherichia coli (E. coli). Sudan II and IV binding to lecithin liposomes and live E. coli corresponds to the Langmuir adsorption isotherm. In the Sudan red compounds – lecithin liposome solutions, the binding ratio of Sudan II to lecithin is 1/31 and that of Sudan IV to 1/314. The binding constant of the Sudan II-lecithin complex is 1.75 × 104 and that of the Sudan IV-lecithin complex 2.92 × 105. Besides, the influences of pH, electrolyte and temperature were investigated and analyzed quantitatively. In the Sudan red compounds – E.coli mixture, the binding ratios of Sudan II and Sudan IV to E.coli membrane phospholipid are 1/29 and 1/114. The binding constants of the Sudan II – and Sudan IV- E.coli membrane phospholipid complexes are 1.86 × 104 and 6.02 × 104. Over 60% of Sudan II and 75% of Sudan IV penetrated into E.coli, in which 90% of them remained in the E.coli membrane. Conclusion Experiments of Sudan II and IV binding to lecithin liposomes and live E. coli indicates that amphiphilic compounds may besequestered in thelecithin liposomes and membrane phospholipid bilayer according to the Langmuir adsorption law. Penetration into the cytosol was impeded and inhibited for Sudan red compounds. It is possible for such compounds themselves (excluding their metabolites and by-products)not result directly in terminal toxicity. Therefore, membrane toxicity could be manifested as membrane blocking and membrane expansion. The method established here may be useful for evaluating the interaction of toxins with membranes. PMID:17389047

IgG red blood cell autoantibodies in autoimmune hemolytic anemia bind to epitopes on red blood cell membrane band 3 glycoprotein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Victoria, E.J.; Pierce, S.W.; Branks, M.J.

1990-01-01

Red blood cell (RBC) autoantibodies from patients with IgG warm-type autoimmune hemolytic anemia were labeled with iodine 125 and their RBC binding behavior characterized. Epitope-bearing RBC membrane polypeptides were identified after autoantibody immunoprecipitation of labeled membranes and immunoblotting. Immunoaffinity isolation of labeled membrane proteins with 12 different IgG hemolytic autoantibodies with protein A-agarose revealed a major polypeptide at Mr 95 to 110 kd, which coelectrophoresed on sodium dodecylsulfate-polyacrylamide gel electrophoresis with a membrane component isolated with sheep IgG anti-band 3. Immunoprecipitation studies with chymotrypsinized RBCs resulted in the recovery of two labeled membrane polypeptides with molecular weights characteristically resulting frommore » the chymotryptic fragmentation of band 3. Immunoblotting with sheep IgG anti-band 3 of the immunoprecipitated polypeptides confirmed that hemolytic autoantibody binding led to recovery of band 3 or its fragments. Two 125I-labeled IgG hemolytic autoantibodies showed binding behavior consistent with epitope localization on band 3. The labeled RBC autoantibodies bound immunospecifically to all types of human RBC tested, including those of rare Rh type (Rh-null, D--) at a site density of approximately 10(6) per RBC. The 125I-IgG in two labeled autoantibodies was 84% and 92% adsorbable by human and higher nonhuman primate RBCs. Antigen-negative animal RBC bound less than 10%, consistent with immunospecific RBC binding. IgG-1 was the major subclass in five autoantibodies tested; one of six fixed complement; and autoantibody IgG appeared polyclonal by isoelectric focusing. We conclude that IgG eluted from RBCs of patients with autoimmune hemolytic anemia consists predominantly of a single totally RBC-adsorbable antibody population that binds to antigenic determinants on band 3.« less

Evaluation of water binding, seed coat permeability and germination characteristics of wheat seeds equilibrated at different relative humidities.

PubMed

Chatterjee, Nabamita; Nagarajan, Shantha

2006-08-01

The relative binding of seed water and seed coat membrane stability were measured in two contrasting wheat (Triticum aestivum L) varieties, HDR 77 (drought-tolerant) and HD 2009 (susceptible) using seed water sorption isotherms, electrical conductivity (EC) of leachates and desorption-absorption isotherms. Analysis of sorption isotherm at 25 degrees C showed that the seeds of HDR 77 had significantly higher number of strong binding sites, with correspondingly greater amount of seed water as strongly bound water, as compared to HD 2009. Total number of binding sites was also higher in HDR 77 than HD 2009, which explained the better desiccation tolerance and higher capacity to bind water in seeds of HDR 77. EC of seed leachate in both varieties did not change with respect to change in equilibrium relative humidity (RII), indicating the general seed coat membrane stability of wheat seeds. However, absolute conductivity values were higher for HD 2009. showing its relatively porous seed coat membrane. Significantly lower area enclosed by the desorption-absorption isotherm loop in HDR 77, as compared to HD 2009 also indicated the greater membrane integrity of HDR 77. Germination and seedling vigour of HD 2009 were reduced when equilibrated over very low and very high RH. In contrast, germination and vigour in HDR 77 were maintained high, except at very high RH, indicating again its desiccation tolerance. Thus, the study demonstrated the relative drought tolerance of HDR 77, on the basis of seed water-binding characteristics and seed membrane stability. Seed membrane stability as measured by seed leachate conductivity or as area under dehydration-rehydration loop may be used as a preliminary screening test for drought tolerance in wheat.

Chemotherapy drugs form ion pores in membranes due to physical interactions with lipids.

PubMed

Ashrafuzzaman, Mohammad; Tseng, Chih-Yuan; Duszyk, Marek; Tuszynski, Jack A

2012-12-01

We demonstrate the effects on membrane of the tubulin-binding chemotherapy drugs: thiocolchicoside and taxol. Electrophysiology recordings across lipid membranes in aqueous phases containing drugs were used to investigate the drug effects on membrane conductance. Molecular dynamics simulation of the chemotherapy drug-lipid complexes was used to elucidate the mechanism at an atomistic level. Both drugs are observed to induce stable ion-flowing pores across membranes. Discrete pore current-time plots exhibit triangular conductance events in contrast to rectangular ones found for ion channels. Molecular dynamics simulations indicate that drugs and lipids experience electrostatic and van der Waals interactions for short periods of time when found within each other's proximity. The energies from these two interactions are found to be similar to the energies derived theoretically using the screened Coulomb and the van der Waals interactions between peptides and lipids due to mainly their charge properties while forming peptide-induced ion channels in lipid bilayers. Experimental and in silico studies together suggest that the chemotherapy drugs induce ion pores inside lipid membranes due to drug-lipid physical interactions. The findings reveal cytotoxic effects of drugs on the cell membrane, which may aid in novel drug development for treatment of cancer and other diseases. © 2012 John Wiley & Sons A/S.

A 115 kDa calmodulin-binding protein is located in rat liver endosome fractions.

PubMed Central

Enrich, C; Bachs, O; Evans, W H

1988-01-01

The distribution of calmodulin-binding polypeptides in various rat liver subcellular fractions was investigated. Plasma-membrane, endosome, Golgi and lysosome fractions were prepared by established procedures. The calmodulin-binding polypeptides present in the subcellular fractions were identified by using an overlay technique after transfer from gels to nitrocellulose sheets. Distinctive populations of calmodulin-binding polypeptides were present in all the fractions examined except lysosomes. A major 115 kDa calmodulin-binding polypeptide of pI 4.3 was located to the endosome subfractions, and it emerges as a candidate endosome-specific protein. Partitioning of endosome fractions between aqueous and Triton X-114 phases indicated that the calmodulin-binding polypeptide was hydrophobic. Major calmodulin-binding polypeptides of 140 and 240 kDa and minor polypeptides of 40-60 kDa were present in plasma membranes. The distribution of calmodulin in the various endosome and plasma-membrane fractions was also analysed, and the results indicated that the amounts were high compared with those in the cytosol. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:3214436

Specific receptors for epidermal growth factor in rat intestinal microvillus membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thompson, J.F.

Epidermal growth factor (EGF) is present in high concentrations in milk, salivary, and pancreaticobiliary secretions. EGF, delivered to the intestinal lumen by these fluids, appears to influence intestinal proliferation. Because EGF exerts its mitogenic effect through binding to specific membrane-bound receptors, binding studies of {sup 125}I-labeled EGF to purified microvillus membrane (MVM) preparations fetal, newborn, and adult rat small intestine were performed. Using the membrane filter technique, binding of {sup 125}I-EGF to adult MVM was specific, saturable, and reversible. Adult and fetal MVM binding was rapid and reached a plateau after 30 min at both 20 and 37{degree}C. No bindingmore » was detected at 4{degree}C. Specific binding increased linearly from 0 to 75 {mu}g MVM protein. Scatchard analysis revealed a single class of receptors in fetal and adult MVM with an association constant of 1.0 {+-} 0.35 {times} 10{sup 9} and 2.3 {+-} 1.6 {times} 10{sup 9} M{sup {minus}1}, respectively. Binding capacity was 435.0 {+-} 89 and 97.7 {+-} 41.3 fmol {sup 125}I-EGF bound/mg MVM protein for fetal and adult MVM, respectively. Newborn MVM binding was negligible. After binding, cross-linking utilizing disuccinimidyl suberate, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography revealed a 170-kDa receptor. These data demonstrate specific receptors for EGF on MVM of rat small intestine and, thus, suggest a mechanism for the intraluminal regulation of enterocyte proliferation by EGF.« less

L-phenylalanyl-L-glutamate-stimulated, chloride-dependent glutamate binding represents glutamate sequestration mediated by an exchange system.

PubMed

Kessler, M; Petersen, G; Vu, H M; Baudry, M; Lynch, G

1987-04-01

Stimulation of glutamate binding by the dipeptide L-phenylalanyl-L-glutamate (Phe-Glu) was inhibited by the peptidase inhibitor bestatin, suggesting that the stimulation was caused by glutamate liberated from the dipeptide and not by the dipeptide itself. It further suggests that this form of glutamate binding should be reinterpreted as glutamate sequestration and that stimulation of binding both by dipeptides and after preincubation with high concentrations of glutamate is likely to be due to counterflow accumulation. Several other criteria indicate that most of glutamate binding stimulated by chloride represents glutamate sequestration: Binding is reduced when the osmolarity of the incubation medium is increased, when membranes incubated with [3H]glutamate are lysed before filtration, and when membranes are made permeable by transient exposure to saponin. Moreover, dissociation of bound glutamate after a 100-fold dilution of the incubation medium is accelerated about 50 times by the addition of glutamate to the dilution medium. This result would be anomalous if glutamate were bound to a receptor site; it suggests instead that glutamate is transported in and out of membrane vesicles by a transport system that preferentially mediates exchange between internal and external glutamate. Glutamate binding contains a component of glutamate sequestration even when measured in the absence of chloride. Sequestration is adequately abolished only after treating membranes with detergents; even extensive lysis, sonication, and freezing/thawing may be insufficient.

Kinetic Measurements Reveal Enhanced Protein-Protein Interactions at Intercellular Junctions

PubMed Central

Shashikanth, Nitesh; Kisting, Meridith A.; Leckband, Deborah E.

2016-01-01

The binding properties of adhesion proteins are typically quantified from measurements with soluble fragments, under conditions that differ radically from the confined microenvironment of membrane bound proteins in adhesion zones. Using classical cadherin as a model adhesion protein, we tested the postulate that confinement within quasi two-dimensional intercellular gaps exposes weak protein interactions that are not detected in solution binding assays. Micropipette-based measurements of cadherin-mediated, cell-cell binding kinetics identified a unique kinetic signature that reflects both adhesive (trans) bonds between cadherins on opposing cells and lateral (cis) interactions between cadherins on the same cell. In solution, proposed lateral interactions were not detected, even at high cadherin concentrations. Mutations postulated to disrupt lateral cadherin association altered the kinetic signatures, but did not affect the adhesive (trans) binding affinity. Perturbed kinetics further coincided with altered cadherin distributions at junctions, wound healing dynamics, and paracellular permeability. Intercellular binding kinetics thus revealed cadherin interactions that occur within confined, intermembrane gaps but not in solution. Findings further demonstrate the impact of these revealed interactions on the organization and function of intercellular junctions. PMID:27009566

Cell biochemistry studied by single-molecule imaging.

PubMed

Mashanov, G I; Nenasheva, T A; Peckham, M; Molloy, J E

2006-11-01

Over the last decade, there have been remarkable developments in live-cell imaging. We can now readily observe individual protein molecules within living cells and this should contribute to a systems level understanding of biological pathways. Direct observation of single fluorophores enables several types of molecular information to be gathered. Temporal and spatial trajectories enable diffusion constants and binding kinetics to be deduced, while analyses of fluorescence lifetime, intensity, polarization or spectra give chemical and conformational information about molecules in their cellular context. By recording the spatial trajectories of pairs of interacting molecules, formation of larger molecular complexes can be studied. In the future, multicolour and multiparameter imaging of single molecules in live cells will be a powerful analytical tool for systems biology. Here, we discuss measurements of single-molecule mobility and residency at the plasma membrane of live cells. Analysis of diffusional paths at the plasma membrane gives information about its physical properties and measurement of temporal trajectories enables rates of binding and dissociation to be derived. Meanwhile, close scrutiny of individual fluorophore trajectories enables ideas about molecular dimerization and oligomerization related to function to be tested directly.

Molecular Dynamics Methodologies for Probing Cannabinoid Ligand/Receptor Interaction

PubMed Central

Lynch, Diane L.; Hurst, Dow P.; Shore, Derek M.; Pitman, Mike C.; Reggio, Patricia H.

2018-01-01

The cannabinoid type 1 and 2 G-protein-coupled receptors are currently important pharmacological targets with significant drug discovery potential. These receptors have been shown to display functional selectivity or biased agonism, a property currently thought to have substantial therapeutic potential. Although recent advances in crystallization techniques have provided a wealth of structural information about this important class of membrane-embedded proteins, these structures lack dynamical information. In order to fully understand the interplay of structure and function for this important class of proteins, complementary techniques that address the dynamical aspects of their function are required such as NMR as well as a variety of other spectroscopies. Complimentary to these experimental approaches is molecular dynamics, which has been effectively used to help unravel, at the atomic level, the dynamics of ligand binding and activation of these membrane-bound receptors. Here, we discuss and present several representative examples of the application of molecular dynamics simulations to the understanding of the signatures of ligand-binding and -biased signaling at the cannabinoid type 1 and 2 receptors. PMID:28750815

Modulation by clamping: Kv4 and KChIP interactions.

PubMed

Wang, Kewei

2008-10-01

The rapidly inactivating (A-type) potassium channels regulate membrane excitability that defines the fundamental mechanism of neuronal functions such as pain signaling. Cytosolic Kv channel-interacting proteins KChIPs that belong to neuronal calcium sensor (NCS) family of calcium binding EF-hand proteins co-assemble with Kv4 (Shal) alpha subunits to form a native complex that encodes major components of neuronal somatodendritic A-type K+ current, I(SA), in neurons and transient outward current, I(TO), in cardiac myocytes. The specific binding of auxiliary KChIPs to the Kv4 N-terminus results in modulation of gating properties, surface expression and subunit assembly of Kv4 channels. Here, I attempt to emphasize the interaction between KChIPs and Kv4 based on recent progress made in understanding the structure complex in which a single KChIP1 molecule laterally clamps two neighboring Kv4.3 N-termini in a 4:4 manner. Greater insights into molecular mechanism between KChIPs and Kv4 interaction may provide therapeutic potentials of designing compounds aimed at disrupting the protein-protein interaction for treatment of membrane excitability-related disorders.

Analysis of CD44-Hyaluronan Interactions in an Artificial Membrane System

PubMed Central

Wolny, Patricia M.; Banerji, Suneale; Gounou, Céline; Brisson, Alain R.; Day, Anthony J.; Jackson, David G.; Richter, Ralf P.

2010-01-01

CD44 is a major cell surface receptor for the large polydisperse glycosaminoglycan hyaluronan (HA). Binding of the long and flexible HA chains is thought to be stabilized by the multivalent nature of the sugar molecule. In addition, high and low molecular weight forms of HA provoke distinct proinflammatory and anti-inflammatory effects upon binding to CD44 and can deliver either proliferative or antiproliferative signals in appropriate cell types. Despite the importance of such interactions, however, neither the stoichiometry of multivalent HA binding at the cell surface nor the molecular basis for functional distinction between different HA size categories is understood. Here we report on the design of a supported lipid bilayer system that permits quantitative analysis of multivalent binding through presentation of CD44 in a stable, natively oriented manner and at controlled density. Using this system in combination with biophysical techniques, we show that the amount of HA binding to bilayers that are densely coated with CD44 increases as a function of HA size, with half-maximal saturation at ∼30 kDa. Moreover, reversible binding was confined to the smaller HA species (molecular weight of ≤10 kDa), whereas the interaction was essentially irreversible with larger polymers. The amount of bound HA decreased with decreasing receptor surface density, but the stability of binding was not affected. From a physico-chemical perspective, the binding properties of HA share many similarities with the typical behavior of a flexible polymer as it adsorbs onto a homogeneously attractive surface. These findings provide new insight into the multivalent nature of CD44-HA interactions and suggest a molecular basis for the distinct biological properties of different size fractions of hyaluronan. PMID:20663884

The Human Metapneumovirus Small Hydrophobic Protein Has Properties Consistent with Those of a Viroporin and Can Modulate Viral Fusogenic Activity

PubMed Central

Masante, Cyril; El Najjar, Farah; Chang, Andres; Jones, Angela; Moncman, Carole L.

2014-01-01

ABSTRACT Human metapneumovirus (HMPV) encodes three glycoproteins: the glycoprotein, which plays a role in glycosaminoglycan binding, the fusion (F) protein, which is necessary and sufficient for both viral binding to the target cell and fusion between the cellular plasma membrane and the viral membrane, and the small hydrophobic (SH) protein, whose function is unclear. The SH protein of the closely related respiratory syncytial virus has been suggested to function as a viroporin, as it forms oligomeric structures consistent with a pore and alters membrane permeability. Our analysis indicates that both the full-length HMPV SH protein and the isolated SH protein transmembrane domain can associate into higher-order oligomers. In addition, HMPV SH expression resulted in increases in permeability to hygromycin B and alteration of subcellular localization of a fluorescent dye, indicating that SH affects membrane permeability. These results suggest that the HMPV SH protein has several characteristics consistent with a putative viroporin. Interestingly, we also report that expression of the HMPV SH protein can significantly decrease HMPV F protein-promoted membrane fusion activity, with the SH extracellular domain and transmembrane domain playing a key role in this inhibition. These results suggest that the HMPV SH protein could regulate both membrane permeability and fusion protein function during viral infection. IMPORTANCE Human metapneumovirus (HMPV), first identified in 2001, is a causative agent of severe respiratory tract disease worldwide. The small hydrophobic (SH) protein is one of three glycoproteins encoded by all strains of HMPV, but the function of the HMPV SH protein is unknown. We have determined that the HMPV SH protein can alter the permeability of cellular membranes, suggesting that HMPV SH is a member of a class of proteins termed viroporins, which modulate membrane permeability to facilitate critical steps in a viral life cycle. We also demonstrated that HMPV SH can inhibit the membrane fusion function of the HMPV fusion protein. This work suggests that the HMPV SH protein has several functions, though the steps in the HMPV life cycle impacted by these functions remain to be clarified. PMID:24672047

Southern blotting.

PubMed

Brown, T

2001-05-01

Southern blotting is the transfer of DNA fragments from an electrophoresis gel to a membrane support (the properties and advantages of the different types of membrane, transfer buffer, and transfer method are discussed in detail), resulting in immobilization of the DNA fragments, so the membrane carries a semipermanent reproduction of the banding pattern of the gel. After immobilization, the DNA can be subjected to hybridization analysis, enabling bands with sequence similarity to a labeled probe to be identified. This appendix describes Southern blotting via upward capillary transfer of DNA from an agarose gel onto a nylon or nitrocellulose membrane, using a high-salt transfer buffer to promote binding of DNA to the membrane. With the high-salt buffer, the DNA becomes bound to the membrane during transfer but not permanently immobilized. Immobilization is achieved by UV irradiation (for nylon) or baking (for nitrocellulose). A Support Protocol describes how to calibrate a UV transilluminator for optimal UV irradiation of a nylon membrane. An alternate protocol details transfer using nylon membranes and an alkaline buffer, and is primarily used with positively charged nylon membranes. The advantage of this combination is that no post-transfer immobilization step is required, as the positively charged membrane binds DNA irreversibly under alkaline transfer conditions. The method can also be used with neutral nylon membranes but less DNA will be retained. A second alternate protocol describes a transfer method based on a different transfer-stack setup. The traditional method of upward capillary transfer of DNA from gel to membrane described in the first basic and alternate protocols has certain disadvantages, notably the fact that the gel can become crushed by the weighted filter papers and paper towels that are laid on top of it. This slows down the blotting process and may reduce the amount of DNA that can be transferred. The downward capillary method described in the second alternate protocol is therefore more rapid than the basic protocol and can result in more complete transfer. Although the ease and reliability of capillary transfer methods makes this far and away the most popular system for Southern blotting with agarose gels, it unfortunately does not work with polyacrylamide gels, whose smaller pore size impedes the transverse movement of the DNA molecules. The third alternate protocol describes an electroblotting procedure that is currently the most reliable method for transfer of DNA from a polyacrylamide gel. Dot and slot blotting are also described.

Hybrid In Silico/In Vitro Approaches for the Identification of Functional Cholesterol-Binding Domains in Membrane Proteins.

PubMed

Di Scala, Coralie; Fantini, Jacques

2017-01-01

In eukaryotic cells, cholesterol is an important regulator of a broad range of membrane proteins, including receptors, transporters, and ion channels. Understanding how cholesterol interacts with membrane proteins is a difficult task because structural data of these proteins complexed with cholesterol are scarce. Here, we describe a dual approach based on in silico studies of protein-cholesterol interactions, combined with physico-chemical measurements of protein insertion into cholesterol-containing monolayers. Our algorithm is validated through careful analysis of the effect of key mutations within and outside the predicted cholesterol-binding site. Our method is illustrated by a complete analysis of cholesterol-binding to Alzheimer's β-amyloid peptide, a protein that penetrates the plasma membrane of brain cells through a cholesterol-dependent process.

Hydroxyl functionalized polytriazole-co-polyoxadiazole as substrates for forward osmosis membranes.

PubMed

Duong, Phuoc H H; Chisca, Stefan; Hong, Pei-Ying; Cheng, Hong; Nunes, Suzana P; Chung, Tai-Shung

2015-02-25

Hydroxyl functionalized polytriazole-co-polyoxadiazole (PTA-POD) copolymers have been synthesized and cast as promising highly thermally stable, chemically resistant, and antiorganic/biological fouling porous substrates for the fabrication of thin-film composite (TFC) forward osmosis (FO) membranes. The roles of PTA/POD ratios in the membrane substrates, TFC layers, and FO membrane performance have been investigated. This study demonstrates that the substrate fabricated from the copolymer containing 40 mol % PTA is optimal for the TFC membranes. Compared to the POD-TFC membrane, the 40 mol % PTA-TFC membrane exhibits a remarkable decrease in structural parameter (S) of more than 3.3 times. In addition, the 40 mol % PTA-TFC membrane is characterized by high water fluxes of 24.9 LMH and 47.2 LMH using 1 M NaCl as the draw solution and DI water as the feed under FO and pressure retarded osmosis (PRO) modes, respectively. Compared to a polysulfone (PSU) supported TFC-FO membrane under similar fabrication conditions, the 40% mol PTA-TFC membrane shows better FO performance and enhanced antifouling properties on the support (lower protein binding propensity and improved bacterial inhibition). Moreover, the performance of the 40 mol % PTA supported TFC-FO membrane can be improved to 37.5 LMH (FO mode)/78.4 LMH (PRO mode) and potentially higher by optimizing the support morphology, the TFC formation, and the post-treatment process. Hence, the use of newly developed hydroxyl functionalized polytriazole-co-polyoxadiazole copolymers may open up a new class of material for FO processes.

Tropomyosin modulates erythrocyte membrane stability

PubMed Central

An, Xiuli; Salomao, Marcela; Guo, Xinhua; Gratzer, Walter; Mohandas, Narla

2007-01-01

The ternary complex of spectrin, actin, and 4.1R (human erythrocyte protein 4.1) defines the nodes of the erythrocyte membrane skeletal network and is inseparable from membrane stability under mechanical stress. These junctions also contain tropomyosin (TM) and the other actin-binding proteins, adducin, protein 4.9, tropomodulin, and a small proportion of capZ, the functions of which are poorly defined. Here, we have examined the consequences of selective elimination of TM from the membrane. We have shown that the mechanical stability of the membranes of resealed ghosts devoid of TM is grossly, but reversibly, impaired. That the decreased membrane stability of TM-depleted membranes is the result of destabilization of the ternary complex of the network junctions is demonstrated by the strongly facilitated entry into the junctions in situ of a β-spectrin peptide, containing the actin- and 4.1R-binding sites, after extraction of the TM. The stabilizing effect of TM is highly specific, in that it is only the endogenous isotype, and not the slightly longer muscle TM that can bind to the depleted membranes and restore their mechanical stability. These findings have enabled us identify a function for TM in elevating the mechanical stability of erythrocyte membranes by stabilizing the spectrin-actin-4.1R junctional complex. PMID:17008534

Perfringolysin O Theta Toxin as a Tool to Monitor the Distribution and Inhomogeneity of Cholesterol in Cellular Membranes

PubMed Central

Maekawa, Masashi; Yang, Yanbo; Fairn, Gregory D.

2016-01-01

Cholesterol is an essential structural component of cellular membranes in eukaryotes. Cholesterol in the exofacial leaflet of the plasma membrane is thought to form membrane nanodomains with sphingolipids and specific proteins. Additionally, cholesterol is found in the intracellular membranes of endosomes and has crucial functions in membrane trafficking. Furthermore, cellular cholesterol homeostasis and regulation of de novo synthesis rely on transport via both vesicular and non-vesicular pathways. Thus, the ability to visualize and detect intracellular cholesterol, especially in the plasma membrane, is critical to understanding the complex biology associated with cholesterol and the nanodomains. Perfringolysin O (PFO) theta toxin is one of the toxins secreted by the anaerobic bacteria Clostridium perfringens and this toxin forms pores in the plasma membrane that causes cell lysis. It is well understood that PFO recognizes and binds to cholesterol in the exofacial leaflets of the plasma membrane, and domain 4 of PFO (D4) is sufficient for the binding of cholesterol. Recent studies have taken advantage of this high-affinity cholesterol-binding domain to create a variety of cholesterol biosensors by using a non-toxic PFO or the D4 in isolation. This review highlights the characteristics and usefulness of, and the principal findings related to, these PFO-derived cholesterol biosensors. PMID:27005662

Perfringolysin O Theta Toxin as a Tool to Monitor the Distribution and Inhomogeneity of Cholesterol in Cellular Membranes.

PubMed

Maekawa, Masashi; Yang, Yanbo; Fairn, Gregory D

2016-03-08

Cholesterol is an essential structural component of cellular membranes in eukaryotes. Cholesterol in the exofacial leaflet of the plasma membrane is thought to form membrane nanodomains with sphingolipids and specific proteins. Additionally, cholesterol is found in the intracellular membranes of endosomes and has crucial functions in membrane trafficking. Furthermore, cellular cholesterol homeostasis and regulation of de novo synthesis rely on transport via both vesicular and non-vesicular pathways. Thus, the ability to visualize and detect intracellular cholesterol, especially in the plasma membrane, is critical to understanding the complex biology associated with cholesterol and the nanodomains. Perfringolysin O (PFO) theta toxin is one of the toxins secreted by the anaerobic bacteria Clostridium perfringens and this toxin forms pores in the plasma membrane that causes cell lysis. It is well understood that PFO recognizes and binds to cholesterol in the exofacial leaflets of the plasma membrane, and domain 4 of PFO (D4) is sufficient for the binding of cholesterol. Recent studies have taken advantage of this high-affinity cholesterol-binding domain to create a variety of cholesterol biosensors by using a non-toxic PFO or the D4 in isolation. This review highlights the characteristics and usefulness of, and the principal findings related to, these PFO-derived cholesterol biosensors.

Checking transfer efficiency and equal loading via qualitative optical way in western blotting.

PubMed

Gong, Jun-Hua; Gong, Jian-Ping; Zheng, Kai-Wen

2017-11-01

The ability to determine that successful transfer and equal loading occur prior to using primary antibodies is important. And total protein staining is commonly used to check transfer efficiency and normalization, which play a crucial role in western blotting. Ponceau S and coomassie blue are commonly used, but there are disadvantages reported in recent years. Therefore, we are interested in finding another method, which is cheap, easy and fast. As we know, protein binding region of PVDF membrane is still hydrophilic when carbinol volatilizes, however, the non-protein binding region of PVDF membrane became hydrophobic again. And this different wettability between non-protein binding region and protein binding region of Polyvinylidene difluoride membrane may be used to check transfer efficiency and equal loading in western blotting. Based on the principle above, we describe an optical approach where an experimenter can observe that the proteins have been transferred to the membrane without any staining within minutes. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.