Roles of arginine and lysine residues in the translocation of a cell-penetrating peptide from (13)C, (31)P, and (19)F solid-state NMR.

Cell-penetrating peptides (CPPs) are small cationic peptides that cross the cell membrane while carrying macromolecular cargoes. We use solid-state NMR to investigate the structure and lipid interaction of two cationic residues, Arg(10) and Lys(13), in the CPP penetratin. (13)C chemical shifts indicate that Arg(10) adopts a rigid beta-strand conformation in the liquid-crystalline state of anionic lipid membranes. This behavior contrasts with all other residues observed so far in this peptide, which adopt a dynamic beta-turn conformation with coil-like chemical shifts at physiological temperature. Low-temperature (13)C-(31)P distances between the peptide and the lipid phosphates indicate that both the Arg(10) guanidinium Czeta atom and the Lys(13) Cepsilon atom are close to the lipid (31)P (4.0-4.2 A), proving the existence of charge-charge interaction for both Arg(10) and Lys(13) in the gel-phase membrane. However, since lysine substitution in CPPs is known to weaken their translocation ability, we propose that the low temperature stabilizes interactions of both lysine and arginine with the phosphates, whereas at high temperatures, the lysine-phosphate interaction is much weaker than the arginine-phosphate interaction. This is supported by the unusually high rigidity of the Arg(10) side chain and its beta-strand conformation at high temperatures. The latter is proposed to be important for ion pair formation by allowing close approach of the lipid headgroups to guanidinium side chains. (19)F and (13)C spin diffusion experiments indicate that penetratin is oligomerized into beta-sheets in gel-phase membranes. These solid-state NMR data indicate that guanidinium-phosphate interactions exist in penetratin, and guanidinium groups play a stronger structural role than ammonium groups in the lipid-assisted translocation of CPPs across liquid-crystalline cell membranes.