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...

The membrane-bound conformation of a cell-penetrating peptide, penetratin, is investigated using solid-state NMR spectroscopy. The (13)C chemical shifts of (13)C, (15)N-labeled residues in the peptide indicate a reversible conformational change from beta-sheet at low temperature to coil-like at high temperature. This conformational change occurs for all residues examined between positions 3 and 13, at peptide/lipid molar ratios of 1:15 and 1:30, in membranes with 25-50% anionic lipids, and in both saturated DMPC/DMPG (1,2-dimyristoyl-sn-glycero-3-phosphatidylchloline/1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol) membranes and unsaturated POPC/POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol) membranes. Thus, it is an intrinsic property of penetratin. The coil state of the peptide has C-H order parameters of 0.23-0.52 for C(alpha) and C(beta) sites, indicating that the peptide backbone is unstructured. Moreover, chemical shift anisotropy lineshapes are uniaxially averaged, suggesting that the peptide backbone undergoes uniaxial rotation around the bilayer normal. These observations suggest that the dynamic state of penetratin at high temperature is a structured turn instead of an isotropic random coil. The thermodynamic parameters of this sheet-turn transition are extracted and compared to other membrane peptides reported to exhibit conformational changes. We suggest that the function of this turn conformation may be to reduce hydrophobic interactions with the lipid chains and facilitate penetratin translocation across the bilayer without causing permanent membrane damage.

The orientation of membrane proteins undergoing fast uniaxial rotation around the bilayer normal can be determined without macroscopic alignment. We show that the motionally averaged powder spectra exhibit their 0° frequency, δ // , at the same position as the peak of an aligned sample with the alignment axis parallel to the magnetic field. This equivalence is exploited to determine the orientation of a β-sheet antimicrobial peptide not amenable to macroscopic alignment, using 13 CO and 15 N chemical shifts from powder spectra. This powder sample approach permits orientation determination of naturally membrane-disruptive proteins in diverse environments and under magicangle spinning.

Human α-defensins (HNPs) are immune defense mini-proteins that act by disrupting microbial cell membranes. Elucidating the three-dimensional structures of HNPs in lipid membranes is important for understanding their mechanisms of action. Using solid-state NMR, we have determined the threedimensional structure of HNP-1 in a microcrystalline state outside the lipid membrane, which provides benchmarks for structure determination and comparison with the membrane-bound state. From a suite of 2D and 3D magic-angle spinning experiments, 13 C and 15 N chemical shifts were obtained that yielded torsion angle constraints while inter-residue distances were obtained to restrain the three-dimensional fold. Together, these constraints led to the first high-resolution SSNMR structure of a human defensin. The SSNMR structure has close similarity to the crystal structures of the HNP family, with the exception of the loop region between the first and second β-strands. The difference, which is partially validated by direct torsion angle measurements of selected loop residues, suggests possible conformational variation and flexibility of this segment of the protein, which may regulate HNP interaction with the phospholipid membrane of microbial cells.

Tachyplesin-I (TP-I) is a 17-residue beta-hairpin antimicrobial peptide containing two disulfide bonds. Linear analogs of TP-I where the four Cys residues were replaced by aromatic and aliphatic residues, TPX4, were found to have varying degrees of activities, with the aromatic analogs similarly potent as TP-I. Understanding the different activities of the linear analogs should give insight into the mechanism of action of TP-I. To this end, we have investigated the dynamic structures of the active TPF4 and the inactive TPA4 in bacteria-mimetic anionic POPE/POPG bilayers and compared them with the wild-type TP-I using solid-state NMR spectroscopy. 13C isotropic chemical shifts and backbone (phi, psi) torsion angles indicate that both TPF4 and TPA4 adopt beta-strand conformations without a beta-turn at key residues. 1H spin diffusion from lipid chains to the peptide indicates that the inactive TPA4 binds to the membrane-water interface, similar to the active TP-I. Thus, neither the conformation nor the depth of insertion of the three peptides correlates with their antimicrobial activities. In contrast, the mobility of the three peptides correlates well with their activities: the active TP-I and TPF4 are both highly mobile in the liquid-crystalline phase of the membrane while the inactive TPA4 is completely immobilized. The different mobilities are manifested in the temperature-dependent 13C and 15N spectra, 13C-1H and 15N-1H dipolar couplings and 1H rotating-frame spin-lattice relaxation times. The dynamics of TP-I and TPF4 are both segmental and global. Combined, these data suggest that TP-I and TPF4 disrupt the membrane by large-amplitude motion in the plane of the membrane. The loss of this motion in TPA4 due to aggregation significantly weakens its activity because a higher peptide concentration is required to disturb lipid packing. Thus molecular motion, rather than structure, appears to be the key determinant for the membrane-disruptive activities of tachyplesins.