Showing papers on "Pore forming protein published in 1999"

Stochastic sensing of organic analytes by a pore-forming protein containing a molecular adapter.

Abstract: The detection of organic molecules is important in many areas, including medicine, environmental monitoring and defence1,5. Stochastic sensing is an approach that relies on the observation of individual binding events between analyte molecules and a single receptor6. Engineered transmembrane protein pores are promising sensor elements for stochastic detection6, and in their simplest manifestation they produce a fluctuating binary (‘on/off’) response in the transmembrane electrical current. The frequency of occurrence of the fluctuations reveals the concentration of the analyte, and its identity can be deduced from the characteristic magnitude and/or duration of the fluctuations. Genetically engineered versions of the bacterial pore-forming protein α-haemolysin have been used to identify and quantify divalent metal ions in solution6. But it is not immediately obvious how versatile binding sites for organic ligands might be obtained by engineering of the pore structure. Here we show that stochastic sensing of organic molecules can be procured from α-haemolysin by equipping the channel with an internal, non-covalently bound molecular ‘adapter’ which mediates channel blocking by the analyte. We use cyclodextrins as the adapters because these fit comfortably inside the pore and present a hydrophobic cavity suitable for binding a variety of organic analytes. Moreover, a single sensing element of this sort can be used to analyse a mixture of organic molecules with different binding characteristics. We envisage the use of other adapters, so that the pore could be ‘programmed’ for a range of sensing functions.

Cysteine-scanning mutagenesis of an eukaryotic pore-forming toxin from sea anemone: topology in lipid membranes.

Abstract: Equinatoxin II is a cysteineless pore-forming protein from the sea anemone Actinia equina It readily creates pores in membranes containing sphingomyelin Its topology when bound in lipid membranes has been studied using cysteine-scanning mutagenesis At approximately every tenth residue, a cysteine was introduced Nineteen single cysteine mutants were produced in Escherichia coli and purified The accessibility of the thiol groups in lipid-embedded cysteine mutants was studied by reaction with biotin maleimide Most of the mutants were modified, except those with cysteines at positions 105 and 114 Mutants R144C and S160C were modified only at high concentrations of the probe Similar results were obtained if membrane-bound biotinylated mutants were tested for avidin binding, but in this case three more mutants gave a negative result: S1C, S13C and K43C Furthermore, mutants S1C, S13C, K20C, K43C and S95C reacted with biotin only after insertion into the lipid, suggesting that they were involved in major conformational changes occurring upon membrane binding These results were further confirmed by labeling the mutants with acrylodan, a polarity-sensitive fluorescent probe When labeled mutants were combined with vesicles, the following mutants exhibited blue-shifts, indicating the transfer of acrylodan into a hydrophobic environment: S13C, K20C, S105C, S114C, R120C, R144C and S160C The overall results suggest that at least two regions are embedded within the lipid membrane: the N-terminal 13‐20 region, probably forming an amphiphilic helix, and the tryptophan-rich 105‐120 region Arg144, Ser160 and residues nearby could be involved in making contacts with lipid headgroups The association with the membrane appears to be unique and different from that of bacterial pore-forming proteins and therefore equinatoxin II may serve as a model for eukaryotic channel-forming toxins

Biochemical characteristics of Eiseniapore, a pore-forming protein in the coelomic fluid of earthworms

Abstract: The cytolytic protein Eiseniapore (38 kDa) from coelomic fluid of the earthworm Eisenia fetida functionally requires sphingomyelin as revealed by using mammalian erythrocytes and phospholipid vesicles. The effects of ions, glycoproteins and phospholipids were investigated for the two-step Eiseniapore action mode, binding and pore formation in different assays. Eiseniapore lysis is activated by thiol groups but inhibited by metal ions. Eiseniapore binding to target membranes is inhibited by Eiseniapore-regulating factor, vitronectin, heparin and lysophosphatidylcholine. Ca2+ and Mg2+ were found to be not necessary for membrane binding or lytic activity. Sphingomyelin was essential for Eiseniapore-induced leakage of liposomes. We describe a cytolytic protein/toxin in Eiseniapore which differs from the established classification; it can be activated by thiol groups and is inhibited by sphingomyelin. Electron microscopy of erythrocyte membranes confirmed ring-shaped structures (pores) with a central channel with outer (10 nm) and inner (3 nm) diameters as shown previously [Lange, S., Nusler, F., Kauschke, E., Lutsch, G., Cooper, E.L. & Herrmann, A. (1997) J. Biol. Chem.272, 20 884–20 892] using artificial membranes. Functional evidence of pore formation by Eiseniapore was revealed as protection of lysis by carbohydrates occurred at an effective diameter above 3 nm. From these results, we suggest a plausible explanation for the mechanism by which components of the earthworm’s immune system destroy non-self components.

Interaction of the pore-forming protein equinatoxin II with model lipid membranes: A calorimetric and spectroscopic study.

Abstract: The interactions of equinatoxin II (EqTxII) with zwitterionic (DPPC) and anionic (DPPG) phospholipids and an equimolar mixture of the two phospholipids (DPPC/DPPG) have been investigated by differential scanning calorimetry (DSC), CD-spectropolarimetry, intrinsic emission fluorescence spectroscopy, and ultrasonic velocimetry EqTxII binds to small unilamellar vesicles formed from negatively charged DPPG lipids, causing a marked reduction in the cooperativity and enthalpy of their gel/liquid-crystalline phase transition This transition is completely abolished at a lipid-to-protein ratio, L/P, of 10 For the mixed DPPC/DPPG vesicles, a 2-fold greater lipid-to-protein ratio (L/P = 20) is required to abolish the phase transition, which corresponds to the same negative charge (-10) of lipid molecules per EqTxII molecule The disappearance of the phase transition of the lipids apparently corresponds to the precipitation of the lipid-protein complex, as suggested by our sound velocity measurements Based on the far-UV CD spectra, EqTxII undergoes two structural transitions in the presence of negatively charged vesicles (DPPG) The first transition coincides with the gel/liquid-crystalline phase transition of the lipids, which suggests that the liquid-crystalline form of negatively charged lipids triggers structural changes in EqTxII The second transition involves the formation of alpha-helical structure Based on these observations, we propose that, in addition to electrostatic interactions, hydrophobic interactions play an important role in EqTxII-membrane association

Effect of the hemolytic lectin CEL-III from Holothuroidea Cucumaria echinata on the ANS fluorescence responses in sensitive MDCK and resistant CHO cells.

Abstract: The addition of CEL-III to sensitive MDCK cells preincubated with 8-anilino-1-naphthalenesulfonate (ANS) caused an increase in the fluorescence intensity of the probe. The increase in the ANS fluorescence caused by CEL-III was Ca2+-dependent and strongly inhibited by 0.1 M lactose, indicating that Ca2+-dependent binding of CEL-III to specific carbohydrate receptors on the plasma membrane is responsible for this phenomenon. In contrast, no significant effect of CEL-III on the ANS fluorescence was observed in CHO cells, which are highly resistant to CEL-III cytotoxicity. In MDCK cells, energy transfer from tryptophan residues to bound ANS molecules was observed in the presence of CEL-III, but not in CHO cells. Furthermore, the amount of ANS bound to MDCK cells increased as the concentration of CEL-III increased. Therefore, a simple interpretation is that the CEL-III-induced increase in ANS fluorescence is attributable to an increase of the hydrophobic region in the plasma membrane where ANS could bind. Immunoblotting analysis of proteins from cells treated with CEL-III indicated that CEL-III oligomers were irreversibly bound to the cells, and the amount of oligomer bound to MDCK cells was much greater than that bound to CHO cells under any conditions tested. The oligomerization may be accompanied by an enhancement of the hydrophobicity of CEL-III molecules, which in turn provides new ANS-binding sites. The difference in susceptibility of MDCK and CHO cells to CEL-III cytotoxicity may be due to a difference in oligomerization of bound CEL-III.

Molecular mechanism for pore-formation in lipid membranes by the hemolytic lectin CEL-III from marine invertebrate Cucumaria echinata

Abstract: The pore-forming activity of CEL-III, a Gal/GalNAc specific lectin from the Holothuroidea Cucumaria echinata, was examined using artificial lipid membranes as a model system of erythrocyte membrane. The carboxyfluorescein (CF)-leakage studies clearly indicated that CEL-III induced the formation of pores in the dipalmitoyl phosphatidyl choline (DPPC)-lactosyl ceramide (LacCer) liposomes effectively but not in the DPPC-glucosyl ceramide (GlcCer) liposomes or DPPC liposomes. Such a leakage of CF was strongly inhibited by lactose, a potent inhibitor of CEL-III, suggesting that the leakage is mediated through the specific binding of CEL-III to the carbohydrate chains on the surface of the liposomes. The leakage of CF from the DPPC-lactosyl ceramide liposomes was pH-dependent, and it increased with increasing pH. The immunoblotting analysis and circular dichroism data indicated that upon interaction with liposomes, CEL-III associated to form an oligomer concomitantly with a marked conformational change. Furthermore, channel measurements showed that CEL-III has an ability to form small ion channels in the planar lipid bilayers consisting of diphytanoylphosphatidylcholine and human globoside (Gb4Cer)/LacCer.