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Transportans 67
For cross-linking of transportan to avidin–TRITC or antibodies, the proteins
were modified with a fourfold molar excess of a bifunctional cross-linker succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate
(SMCC) in 0.1 M phosphate
buffer, pH 8.5, for 5 min, as described by Peeters et al. 34 Nonreacted crosslinker
was removed by gel filtration on Sephadex G-15 in 0.1 M phosphate buffer,
pH 6.7, and the SMCC-modified protein was subsequently reacted overnight with
transportan–cysteine using twofold molar excess of peptide.
Studies of the internalization of peptides into cells require a method of visualization,
such as including a label to their sequences. Many commercially available
labels, e.g., fluorescein, rhodamine, biotin, and digoxigenin, can be applied to label
free amino groups in the sequence of peptides. Alternatively, radioactively labeled
peptides can be used to characterize the internalization process quantitatively.
We have used biotinylated peptides because biotinylation can easily be performed
using solid phase peptide synthesis. The internalization of the biotinylated
peptides can be followed using indirect immunofluorescence. This method for visualization
of the biotinyl label includes treatment of cells with biotinyl–peptides,
permeabilization of cells, and subsequent treatment with an avidin or streptavidin–fluorochrome
conjugate. Streptavidin is less basic and is not glycosylated, therefore
showing fewer nonspecific reactions such as interactions with lectins or acidic
groups on proteins or membranes, as compared to avidin.
In our studies, cells were treated with biotinylated peptides and, after permeabilization,
were stained with streptavidin–FITC or avidin–TRITC and visualized under
a fluorescence microscope. Two alternative fixation methods were used. First, cells
were fixed and permeabilized simultaneously with methanol. As both fixing and
permabilization occur in parallel, some of the peptides associated with the plasma
membrane could be redistributed into the cell. The other method used was to fix
cells first with paraformaldehyde and subsequently permeabilize with Hepes
buffer/Triton X-100 mixture. This treatment should exclude the possibility of washing
noninternalized peptides into the cell. However, in our experiments similar results
were obtained with both methods.
3.8 STRUCTURAL ORGANIZATION OF TRANSPORTAN
Circular dichroism studies reveal that, in water, transportan behaves as a random
coil and does not fold into any stable structure. 36 In the presence of SDS micelles,
transportan adopts a secondary structure consisting of 60% helicity. The helix is
localized in mastoparan part and galanin-derived N-terminal part is less structured.
However, both parts are almost buried in the SDS micelles, leaving only the central
connecting part of transportan clearly exposed to the solution, as shown by NMR
studies. Considering these data, the interaction of transportan with membranes can
be hypothesized. The mastoparan helix is inserted deeply into lipid bilayer with its
axis perpendicular to the membrane interface; the connecting segment, containing
the Lys residue where the labels and cargoes are coupled, protrudes from the
membrane. The N terminus is residing partly in the membrane interface and is buried
partly in the membrane. 36