Conformational and topological requirements of cell-permeable peptide function.

Cell-permeable peptide import recently was developed to deliver synthetic peptides into living cells for studying intracellular protein functions. This import process is mediated by an N-terminal carrier sequence which is the hydrophobic region of a signal peptide. In this study, the conformational consequence of the interaction of cell-permeable peptides with different mimetic membrane environments was investigated by circular dichroism analysis. We showed that cell-permeable peptides adopted alpha-helical structures in sodium dodecyl sulfate (SDS) micelles or aqueous trifluoroethanol (TFE). The potency of these peptides in forming helical structures is higher in an amphiphilic environment (SDS) than in a hydrophobic environment (TFE), suggesting that some hydrophilic molecules associated with the cell membrane may be involved in peptide import. We also studied topological requirements of cell-permeable peptide function. We demonstrated that peptides containing the carrier sequence in their C-termini can also be imported into cells efficiently. This important discovery can avoid repetitious synthesis of the membrane-translocating sequence for peptides with different functional cargoes and is potentially useful for developing a cell-permeable peptide library. Finally, we showed that, when a retro version of the carrier sequence was used, the peptide lost its translocating ability despite retaining a high content of alpha-helical structure in mimetic membrane environments. This suggests that the propensity of peptides to adopt a helical conformation is required but not sufficient for cellular import and that other structural factors such as the side-chain topology of the carrier sequence are also important. Our studies together contribute to the more rational design of useful cell-permeable peptides.