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  • 1
    Electronic Resource
    Electronic Resource
    Structure-function relationships in diphtheria toxin channels: III. Residues which affect the cis pH dependence of channel conductance (1994)
    Springer
    The journal of membrane biology 137 (1994), S. 45-57 
    Details
    ISSN: 1432-1424
    Keywords: Diphtheria toxin ; Site-directed mutagenesis ; Planar lipid bilayers ; Single channel conductance ; Ion selectivity ; pH dependence
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract The conductance of channels formed by diphtheria toxin (DT) in lipid bilayer membranes depends strongly on pH. We have previously shown that a 61 amino acid region of the protein, denoted TH8-9, is sufficient to form channels having the same pH-dependent conductance properties as those of whole toxin channels. One residue in this region, Aspartate 352, is responsible for all the dependence of single channel conductance on trans pH, whereas another, Glutamate 349, has no effect. Here, we report that of the seven remaining charged residues in the TH8-9 region, mutations altering the charge on H322, H323, H372, and R377 have minimal effects on single channel conductance; mutations of Glutamates 326, 327, or 362, however, significantly affect single channel conductance as well as its dependence on cis pH. Moreover, Glutamate 362 is titratable from both the cis and trans sides of the membrane, suggesting that this residue lies within the channel; it is more accessible, however, to cis than to trans protons. These results are consistent with the membrane-spanning topology previously proposed for the TH8-9 region, and suggest a geometric model for the DT channel.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00234997
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  • 2
    Electronic Resource
    Electronic Resource
    Structure function relationships in diphtheria toxin channels: II. A residue responsible for the channel's dependence on trans pH (1994)
    Springer
    The journal of membrane biology 137 (1994), S. 29-44 
    Details
    ISSN: 1432-1424
    Keywords: Diphtheria toxin ; Site-directed mutagenesis ; Planar lipid bilayers ; Single channel conductance ; Ion selectivity ; pH dependence
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Ion-conducting channels formed in lipid bilayers by diphtheria toxin are highly pH dependent. Among other properties, the channel's single channel conductance and selectivity depend on proton concentrations on either side of the membrane. We have previously shown that a 61 amino acid fragment of DT is sufficient to form a channel having the same pH-dependent single channel properties as that of the intact toxin. This region corresponds to an a-helical hairpin in the recently published crystal structure of DT in solution; the hairpin contains two α-helices, each long enough to span a membrane, connected by a loop of about nine residues. This paper reports on the single channel effects of mutations which alter the two negatively charged residues in this loop. Changing Glutamate 349 to neutral glutamine or to positive lysine has no effect on the DT channel's single channel conductance or selectivity. In contrast, mutations of Aspartate 352 to neutral asparagine (DT-D352N) or positive lysine (DT-D352K) cause progressive reductions in single channel conductance at pH 5.3 cis/7.2 trans (in 1 m KCl), consistent with this group interacting electrostatically with ions in the channel. The cation selectivity of these mutant channels is also reduced from that of wild-type channels, a direction consistent with residue 352 influencing permeant ions via electrostatic forces. When both sides of the membrane are at pH 4, the conductance difference between wild-type and DT-D352N channels is minimal, suggesting that Asp 352 (in the wild type) is neutral at this pH. Differences observed between wild-type and DT-D352N channels at pH 4.0 cis/7.2 trans (with a high concentration of permeant buffer in the cis compartment) imply that residue 352 is on or near the trans side of the membrane. Comparing the conductances of wild-type and DT-D352K channels at large (cis) positive voltages supports this conclusion. The trans location of position 352 severely constrains the number of possible membrane topologies for this region.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00234996
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  • 3
    Electronic Resource
    Electronic Resource
    Structure-function relationships in diphtheria toxin channels: I. Determining a minimal channel-forming domain (1994)
    Springer
    The journal of membrane biology 137 (1994), S. 17-28 
    Details
    ISSN: 1432-1424
    Keywords: Diphtheria toxin ; Site-directed mutagenesis ; Planar lipid bilayers ; Ion channels ; T-domain ; Channel-forming peptides
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Diphtheria Toxin (DT) is a 535 amino acid exotoxin, whose active form consists of two polypeptide chains linked by an interchain disulphide bond. DT's N-terminal A fragment kills cells by enzymatically inactivating their protein synthetic machinery; its C terminal B chain is required for the binding of toxin to sensitive cells and for the translocation of the A fragment into the cytosol. This B fragment, consisting of its N-terminal T domain (amino acids 191–386) and its C-terminal R domain (amino acids 387–535) is responsible for the ion-conducting channels formed by DT in lipid bilayers and cellular plasma membranes. To further delineate the channel-forming region of DT, we studied channels formed by deletion mutants of DT in lipid bilayer membranes under several pH conditions. Channels formed by mutants containing only the T domain (i.e., lacking the A fragment and/or the R domain), as well as those formed by mutants replacing the R domain with Interleukin-2 (Il–2), have single channel conductances and selectivities essentially identical to those of channels formed by wild-type DT. Furthermore, deleting the N-terminal 118 amino acids of the T domain also has minimal effect on the single channel conductance and selectivity of the mutant channels. Together, these data identify a 61 amino acid stretch of the T domain, corresponding to the region which includes α-helices TH8 and TH9 in the crystal structure of DT, as the channel-forming region of the toxin.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00234995
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