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crc press - E-Lib FK UWKS

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Transportans 67<br />

For cross-linking of transportan to avidin–TRITC or antibodies, the proteins<br />

were modified with a fourfold molar excess of a bifunctional cross-linker succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate<br />

(SMCC) in 0.1 M phosphate<br />

buffer, pH 8.5, for 5 min, as described by Peeters et al. 34 Nonreacted crosslinker<br />

was removed by gel filtration on Sephadex G-15 in 0.1 M phosphate buffer,<br />

pH 6.7, and the SMCC-modified protein was subsequently reacted overnight with<br />

transportan–cysteine using twofold molar excess of peptide.<br />

Studies of the internalization of peptides into cells require a method of visualization,<br />

such as including a label to their sequences. Many commercially available<br />

labels, e.g., fluorescein, rhodamine, biotin, and digoxigenin, can be applied to label<br />

free amino groups in the sequence of peptides. Alternatively, radioactively labeled<br />

peptides can be used to characterize the internalization process quantitatively.<br />

We have used biotinylated peptides because biotinylation can easily be performed<br />

using solid phase peptide synthesis. The internalization of the biotinylated<br />

peptides can be followed using indirect immunofluorescence. This method for visualization<br />

of the biotinyl label includes treatment of cells with biotinyl–peptides,<br />

permeabilization of cells, and subsequent treatment with an avidin or streptavidin–fluorochrome<br />

conjugate. Streptavidin is less basic and is not glycosylated, therefore<br />

showing fewer nonspecific reactions such as interactions with lectins or acidic<br />

groups on proteins or membranes, as compared to avidin.<br />

In our studies, cells were treated with biotinylated peptides and, after permeabilization,<br />

were stained with streptavidin–FITC or avidin–TRITC and visualized under<br />

a fluorescence microscope. Two alternative fixation methods were used. First, cells<br />

were fixed and permeabilized simultaneously with methanol. As both fixing and<br />

permabilization occur in parallel, some of the peptides associated with the plasma<br />

membrane could be redistributed into the cell. The other method used was to fix<br />

cells first with paraformaldehyde and subsequently permeabilize with Hepes<br />

buffer/Triton X-100 mixture. This treatment should exclude the possibility of washing<br />

noninternalized peptides into the cell. However, in our experiments similar results<br />

were obtained with both methods.<br />

3.8 STRUCTURAL ORGANIZATION OF TRANSPORTAN<br />

Circular dichroism studies reveal that, in water, transportan behaves as a random<br />

coil and does not fold into any stable structure. 36 In the presence of SDS micelles,<br />

transportan adopts a secondary structure consisting of 60% helicity. The helix is<br />

localized in mastoparan part and galanin-derived N-terminal part is less structured.<br />

However, both parts are almost buried in the SDS micelles, leaving only the central<br />

connecting part of transportan clearly exposed to the solution, as shown by NMR<br />

studies. Considering these data, the interaction of transportan with membranes can<br />

be hypothesized. The mastoparan helix is inserted deeply into lipid bilayer with its<br />

axis perpendicular to the membrane interface; the connecting segment, containing<br />

the Lys residue where the labels and cargoes are coupled, protrudes from the<br />

membrane. The N terminus is residing partly in the membrane interface and is buried<br />

partly in the membrane. 36

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