Roles of Arginine and Lysine Residues in the Translocation of a Cell-Penetrating Peptide from¹³C,³¹P, and¹⁹F Solid-State NMR

Abstract

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 β-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 β-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 Cζ and the Lys 13 Cε lie in close proximity to the lipid 31 P (4.0 -4.2 Å), 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 are known to reduce their translocation ability, we propose that low temperature stabilizes both lysine and arginine interactions with the phosphates, whereas at high temperature the lysine-phosphate interaction is much weaker than the argininephosphate interaction. This is supported by the unusually high rigidity of the Arg 10 sidechain and its β-strand conformation at high temperature. The latter is proposed to be important for ion pair formation by allowing close approach of the lipid headgroups to guanidinium sidechains. 19 F and 13 C spin diffusion experiments indicate that penetratin is oligomerized into β-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. Keywordscell-penetrating peptide; lipid bilayer; guanidinium-phosphate interaction; 13 C-31 P REDOR; arginine; lysine Cell-penetrating peptides (CPP) are arginine-and lysine-rich cationic peptides that can readily enter cells not only by themselves but also carrying other macromolecular cargos (1-3). Thus they are promising drug-delivery molecules. Many studies have established that the intracellular entry of CPPs is related to their strong affinity to lipid bilayers (4). The lipid *Corresponding author: Mei Hong, Department of Chemistry, Iowa State University, Ames, IA 50011. Tel: 515-294-3521, Fax: 515-294-0105, E-mail: mhong@iastate.edu. Supporting information available 13 C chemical shift assignments of Arg 10 -labeled penetratin in POPC/POPG membranes and RMSD analyses of REDOR data are provided. This supplemental material is free of charge online at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 June 2. membrane can be the plasma membrane of the cell or the endosomal membrane from which CPPs must escape after endocytosis (5). The fundamental biophysical question of interest is how these highly cationic peptides cross th...

Structure and dynamics of cationic membrane peptides and proteins: Insights from solid‐state NMR

“…107 However, among five examined residues, one residue, Arg10, retained the b-strand torsion angles at high temperature and exhibited a nearly rigid-limit CaHa order parameter (S CH ¼ 0.92). 108 This difference suggests that Arg molecules, due to their interactions with lipid phosphates, may stabilize the conformation of the otherwise coil or turn-rich penetratin.…”

“…Figure 6 summarizes key 13 C-31 P distances measured in the two peptides. 6,108 Penetratin exhibits short side chain 13 C-31 P distances of 4.2 and 4.0 Å from Arg10 Cf and Lys13 Ce, respectively, whereas the neutral side chains of Ile3 has much longer distances. 108 Therefore, short 13 C-31 P distances are specific to cationic side chains due to charge neutralization and H-bonding.…”

Peptides as Promising Non-Viral Vectors for Gene Therapy

“…At the same time, it transforms itself into a more non-polar compound either due to charge neutralisation (Su et al, 2009) or hydrogen bonding (Rothbard et al, 2004) with the anionic membrane proteins. The peptide then partitions into the hydrophobic lipid bilayer (Rothbard et al, 2002) and translocates through the membrane in a process that is driven by the voltage potential across membrane (Rothbard et al, 2004) and/or the need to relieve membrane curvature stress caused by the mass imbalance in the initial step (Su et al, 2009). In stark contrast, Richard and colleagues reported that the uptake of tat 48-59, in both its free and PNA-conjugated forms were sensitive to low temperature and sodium azide, indicative of classical endocytosis (Richard et al, 2003).…”

Reducible chimeric polypeptide consisting of octa-d-arginine and tetra-l-histidine peptides as an efficient gene delivery vector

“…14,15 Although Won et al [16][17][18] reported reducible arginine polypeptides, they did not include the buffering moieties in these polypeptides. More importantly, all these homogeneous reducible polypeptides were prepared using fixed compositions of monomers.…”