Abstract
The homeodomain, the DNA‐binding domain of Antennapedia homeoprotein, is composed of three α‐helices and one β‐turn between helices II and III. Its third helix from the N‐terminal (helix III) can translocate through the cell membrane into the nucleus and can be used as an intracellular vehicle for the delivery of oligopeptides and oligonucleotides. To the best of our knowledge, this helix III, called penetratin, which consists of 16 amino acids, is internalized by cells in a specific, non‐receptor‐mediated manner. For a better understanding of the mechanism of the transfer, the structure of penetratin was examined in both extracellular matrix‐mimetic and membrane‐mimetic environments; ^1^H‐NMR and CD spectroscopic measurements were performed in mixtures of TFE/water with different ratios. The molecular conformations of two analogue peptides [(6,14‐Phe)‐penetratin and a 12 amino acid penetratin derivative (peptide 3)] were also studied. An atomic level comprehensive analysis of penetratin and its two analogues was performed. In a membrane‐mimetic solvent system (TFEd~2~/water = 9 : 1), on the basis of 553 distance restraints, the 4–12 region of penetratin exhibits a bent, irregular helical structure on NMR examination. Interactions between hydrophobic amino acid residues in conjunction with H‐bonds stabilize the secondary structure of the molecule. Thus, both derivatives adopt a helix‐like conformation. However, while (6,14‐Phe)‐penetratin displays both α‐helical and 3~10~‐helical features, the structure of peptide 3 is predominantly a 3~10~‐helix. Of the three peptides, surprisingly (6,14‐Phe)‐penetratin has the largest helical content. An increase in the polarity of the molecular environment gradually disintegrates these helix‐like secondary structures. In a highly aqueous molecular system (TFEd~2~/water = 1 : 9), the fast exchange of multiple conformers leads to too few distance restraints being extracted, therefore the NMR structures can no longer be determined. The NMR data show that only short‐range order can be traced in these peptides. Under these conditions, the molecules adopt nascent helix‐like structures. On the other hand, CD spectra could be recorded at any TFE/water ratio and the conformational interconversion could therefore be monitored as a function of the polarity of the molecular environment. The CD data were analysed comprehensively by the quantitative deconvolution method (CCA+). All three penetratin peptides display helical conformational features in a low dielectric medium, with significant differences as a function of their amino acid composition. However, these conformational features are gradually lost during the shift from an apolar to a polar molecular environment. Copyright © 2002 European Peptide Society and John Wiley & Sons, Ltd.