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.
Du
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Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2363, USA.
Yao
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Rojas
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Lin
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YZ
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D002463:Cell Membrane Permeability; D002942:Circular Dichroism; D019084:Fluorescent Antibody Technique, Indirect; D051379:Mice; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation
D010455:Peptides