sodium-dodecyl-sulfate and mastoparan

Transportan is a 27-residue peptide (GWTLN SAGYL LGKIN LKALA ALAKK IL-amide) which has the ability to penetrate into living cells carrying a hydrophilic load. Transportan is a chimeric peptide constructed from the 12 N-terminal residues of galanin in the N-terminus with the 14-residue sequence of mastoparan in the C-terminus and a connecting lysine. Circular dichroism studies of transportan and mastoparan show that both peptides have close to random coil secondary structure in water. Sodium dodecyl sulfate (SDS) micelles induce 60% helix in transportan and 75% helix in mastoparan. The 600 MHz (1)H NMR studies of secondary structure in SDS micelles confirm the helix in mastoparan and show that in transportan the helix is localized to the mastoparan part. The less structured N-terminus of transportan has a secondary structure similar to that of the same sequence in galanin [Ohman, A., et al. (1998) Biochemistry 37, 9169-9178]. The position of mastoparan and transportan relative to the SDS micelle surface was studied by adding spin-labeled 5-doxyl- or 12-doxyl-stearic acid or Mn2+ to the peptide/micelle system. The combined results show that the peptides are for the most part buried in the SDS micelles. Only the C-terminal parts of both peptides and the central segment connecting the two parts of transportan are clearly surface exposed. For mastoparan, the secondary chemical shifts of the amide protons were found to vary periodically and display a pattern almost identical to those reported for mastoparan in phospholipid bicelles [Vold, R., et al. (1997) J. Biomol. NMR 9, 329-335], indicating similar structures and interactions in the two membrane-mimicking environments.

Topics: Amino Acid Sequence; Animals; Biological Transport, Active; Cell Membrane; Circular Dichroism; Cyclic N-Oxides; Drug Carriers; Galanin; Intercellular Signaling Peptides and Proteins; Micelles; Molecular Sequence Data; Nuclear Magnetic Resonance, Biomolecular; Peptides; Protein Structure, Secondary; Recombinant Fusion Proteins; Sodium Dodecyl Sulfate; Spin Labels; Wasp Venoms

The influence of sodium dodecyl sulfate (SDS) on ADP-ribosylation by Clostridium botulinum C3 exoenzyme (C3) was studied. SDS increased the ADP-ribosylation of recombinant rhoA and human platelet cytosolic proteins maximally at 0.01% whereas higher concentrations of the detergent (> 0.01%) inhibited the ADP-ribosylation. In contrast, ADP-ribosylation of human platelet membranes and of recombinant rhoB was inhibited by the detergent. The Km for NAD of the ADP-ribosylation of rhoA was decreased by SDS from about 10 to 0.6 microM. Whereas in the absence of SDS, the C3-induced ADP-ribosylation of recombinant rhoA is not affected by the amphiphilic wasp venom mastoparan, in the presence of SDS (0.01%) mastoparan (100 microM) inhibited the ADP-ribosylation. C3-associated NAD-glycohydrolase activity was maximally and half-maximally inhibited by 0.1 and 0.013% SDS, respectively. Inhibition of NAD-glycohydrolase activity was reversed by diluting out SDS indicating that C3 was not irreversibly denatured by SDS treatment. SDS (0.01%) completely inhibited the [3H]GTP binding of rhoA whereas the release of previously bound nucleotide was not affected. The data indicate that changes in the lipophilicity of rhoA protein largely affect its ability to serve as a substrate for C3-like ADP-ribosyltransferases.

Topics: ADP Ribose Transferases; Animals; Blood Platelets; Botulinum Toxins; Clostridium botulinum; Dose-Response Relationship, Drug; GTP-Binding Proteins; Humans; Intercellular Signaling Peptides and Proteins; Membrane Proteins; NAD+ Nucleosidase; Peptides; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Recombinant Proteins; rhoA GTP-Binding Protein; rhoB GTP-Binding Protein; Sodium Dodecyl Sulfate; Swine; Wasp Venoms