Tryptophan, more than just an interfacial amino acid in the membrane activity of cationic cell-penetrating and antimicrobial peptides.

Trp is unique among the amino acids since it is involved in many different types of noncovalent interactions such as electrostatic and hydrophobic ones, but also in π-π, π-cation, π-anion and π-ion pair interactions. In membranotropic peptides and proteins, Trp locates preferentially at the water-membrane interface. In antimicrobial or cell-penetrating peptides (AMPs and CPPs respectively), Trp is well-known for its strong role in the capacity of these peptides to interact and affect the membrane organisation of both bacteria and animal cells at the level of the lipid bilayer. This essential amino acid can however be involved in other types of interactions, not only with lipids, but also with other membrane partners, that are crucial to understand the functional roles of membranotropic peptides. This review is focused on this latter less known role of Trp and describes in details, both in qualitative and quantitative ways: (i) the physico-chemical properties of Trp; (ii) its effect in CPP internalisation; (iii) its importance in AMP activity; (iv) its role in the interaction of AMPs with glycoconjugates or lipids in bacteria membranes and the consequences on the activity of the peptides; (v) its role in the interaction of CPPs with negatively charged polysaccharides or lipids of animal membranes and the consequences on the activity of the peptides. We intend to bring highlights of the physico-chemical properties of Trp and describe its extensive possibilities of interactions, not only at the well-known level of the lipid bilayer, but with other less considered cell membrane components, such as carbohydrates and the extracellular matrix. The focus on these interactions will allow the reader to reevaluate reported studies. Altogether, our review gathers dedicated studies to show how unique are Trp properties, which should be taken into account to design future membranotropic peptides with expected antimicrobial or cell-penetrating activity.

A Quantitative Method to Distinguish Cytosolic from Endosome-Trapped Cell-Penetrating Peptides.

Cell-penetrating peptides are known to penetrate cells through endocytosis and translocation. The two pathways are hardly distinguished in current cell assays. We developed a reliable, simple and robust method to distinguish and quantify independently the two routes. The assay requires (DABCYL) 4-(dimethylaminoazo)benzene-4-carboxylic acid- and (CF) carboxyfluorescein-labeled peptides. When the labeled peptide is intact, the fluorescence signal is weak thanks to the dark quenching property of DABCYL. A 10-fold higher fluorescence signal is measured when the labeled peptide is degraded. By referring to a standard fluorescent curve according to the concentration of the hydrolyzed peptide, we have access to the internalized peptide quantity. Therefore, cell lysis after internalization permits to determine the total quantity of intracellular peptide. The molecular state of the internalized peptide (intact or degraded), depends on its location in cells (cytosol vs endo-lysosomes), and can be blocked by boiling cells. This boiling step results indeed in denaturation and inhibition of the cellular enzymes. The advantage of this method is the possibility to quantify translocation at 37 °C and to compare it to the 4 °C condition, where all endocytosis processes are inhibited. We found that ranking of the translocation efficacy is DABCYL-R6-(ϵCF)K≫DABCYL-R4-(ϵCF)K≥CF-R9.

Binding and crossing: Methods for the characterization of membrane-active peptides interactions with membranes at the molecular level.

Antimicrobial and cell-penetrating peptides have been the object of extensive studies for more than 60 years. Initially these two families were studied separately, and more recently parallels have been drawn. These studies have given rise to numerous methodological developments both in terms of observation techniques and membrane models. This review presents some of the most recent original and innovative developments in this field, namely droplet interface bilayers (DIBs), new fluorescence approaches, force measurements, and photolabelling.

Influence of the Dabcyl group on the cellular uptake of cationic peptides: short oligoarginines as efficient cell-penetrating peptides.

Cell-penetrating peptides (CPPs) are promising delivery vehicles. These short peptides can transport wide range of cargos into cells, although their usage has often limitations. One of them is the endosomatic internalisation and thus the vesicular entrapment. Modifications which increases the direct delivery into the cytosol is highly researched area. Among the oligoarginines the longer ones (n > 6) show efficient internalisation and they are well-known members of CPPs. Herein, we describe the modification of tetra- and hexaarginine with (4-((4-(dimethylamino)phenyl)azo)benzoyl) (Dabcyl) group. This chromophore, which is often used in FRET system increased the internalisation of both peptides, and its effect was more outstanding in case of hexaarginine. The modified hexaarginine may enter into cells more effectively than octaarginine, and showed diffuse distribution besides vesicular transport already at low concentration. The attachment of Dabcyl group not only increases the cellular uptake of the cell-penetrating peptides but it may affect the mechanism of their internalisation. Their conjugates with antitumor drugs were studied on different cells and showed antitumor activity.

An Artificial Amphiphilic Peptide Promotes Endocytic Uptake by Inducing Membrane Curvature.

Membrane curvature plays a pivotal role in cellular life, including cellular uptake and membrane trafficking. The modulation of membrane curvature provides a novel means of manipulating cellular events. In this report, we show that a nine-residue amphiphilic peptide (R6W3) stimulates endocytic uptake by inducing membrane curvature. Curvature formation on cell membranes was confirmed by observing the cellular distribution of the curvature-sensing protein amphiphysin fused with a yellow fluorescent protein (Amp-YFP). Dot-like signals of Amp-YFP were visible following the addition of R6W3, suggesting curvature formation in cell membranes, leading to endocytic cup and vesicle formation. The promotion of endocytic uptake was confirmed using the endocytosis marker polydextran. Treatment of cells with R6W3 yielded a 4-fold dextran uptake compared with untreated cells. The amphiphilic helical structure of R6W3 was also crucial for R6W3-stimulated endocytic uptake.

Benzophenone Photoreactivity in a Lipid Bilayer To Probe Peptide/Membrane Interactions: Simple System, Complex Information.

Affinity photo-cross-linking coupled to mass spectrometry, using benzophenone (Bzp)-functionalized peptides, was used to study the noncovalent interactions of cell-penetrating peptides and lipid membranes. Using biomimetic lipid vesicles composed of saturated and unsaturated negatively charged lipids, DMPG (14:0), DPPG (16:0), DOPG (18:1 cis Δ9), 18:1 (trans Δ9) PG, and DLoPG (18:2 cis Δ9, 12), allowed observation of all the classical and less common reactivities of Bzp described in the literature by direct MS analysis: C═C double bond formation on saturated fatty acids, covalent adducts formation via classical C-C bond, and Paternò-Büchi oxetane formation followed or not by fragmentation (retro-Paternò-Büchi) as well as photosensitization of unsaturated lipids leading to lipid dimers. All these reactions can occur concomitantly in a single complex biological system: a membrane-active peptide inserted within a phospholipid bilayer. We also detect oxidation species due to the presence of radical oxygen species. This work represents a noteworthy improvement for the characterization of interacting partners using Bzp photo-cross-linking, and it shows how to exploit in an original way the different reactivities of Bzp in the context of a lipid membrane. We propose an analytical workflow for the interpretation of MS spectra, giving access to information on the CPP/lipid interaction at a molecular level such as depth of insertion or membrane fluidity in the CPP vicinity. An application of this workflow illustrates the role of cholesterol in the CPP/lipids interaction.

Small AntiMicrobial Peptide With in Vivo Activity Against Sepsis.

Antimicrobial peptides (AMPs) are considered as potential therapeutic sources of future antibiotics because of their broad-spectrum activities and alternative mechanisms of action compared to conventional antibiotics. Although AMPs present considerable advantages over conventional antibiotics, their clinical and commercial development still have some limitations, because of their potential toxicity, susceptibility to proteases, and high cost of production. To overcome these drawbacks, the use of peptides mimics is anticipated to avoid the proteolysis, while the identification of minimalist peptide sequences retaining antimicrobial activities could bring a solution for the cost issue. We describe here new polycationic ß-amino acids combining these two properties, that we used to design small dipeptides that appeared to be active against Gram-positive and Gram-negative bacteria, selective against prokaryotic versus mammalian cells, and highly stable in human plasma. Moreover, the in vivo data activity obtained in septic mice reveals that the bacterial killing effect allows the control of the infection and increases the survival rate of cecal ligature and puncture (CLP)-treated mice.

Membrane Crossing and Membranotropic Activity of Cell-Penetrating Peptides: Dangerous Liaisons?

Living organisms have to maintain a stable balance in molecules and ions in the changing environment in which they are living, a process known as homeostasis. At the level of cells, the plasma membrane has a major role in homeostasis, since this hydrophobic film prevents passive diffusion of large and hydrophilic molecules between the extracellular and intracellular milieu. Living organisms have evolved with highly sophisticated transport systems to control exchanges across this barrier: import of nutrients and fuel essential for their survival; recognition of chemical or physical messengers allowing information interchanges with surrounding cells. Besides specialized proteins, endocytosis mechanisms at the level of the lipid bilayer can transport molecules from the outside across the cell membrane, in an energy-dependent manner. The cell membrane is highly heterogeneous in its molecular composition (tens of different lipids, proteins, polysaccharides, and combinations of these) and dynamic with bending, deformation, and elastic properties that depend on the local composition of membrane domains. Many viruses, microorganisms, and toxins exploit the plasma membrane to enter into cells. Chemists develop strategies to target the plasma membrane with molecules capable of circumventing this hydrophobic barrier, in particular to transport and deliver nonpermeable drugs in cells for biotechnological or pharmaceutical purposes. Drug delivery systems are numerous and include lipid-, sugar-, protein-, and peptide-based delivery systems, since these biomolecules generally have good biocompatibility, biodegradability, environmental sustainability, cost effectiveness, and availability. Among those, cell-penetrating peptides (CPPs), reported for the first time in the early 1990s, are attracting major interest not only as potential drug delivery systems but also at the level of fundamental research. It was indeed demonstrated very early that these peptides, which generally correspond to highly cationic sequences, can still cross the cell membrane at 4 °C, a temperature at which all active transport and endocytosis pathways are totally inhibited. Therefore, how these charged hydrophilic peptides cross the hydrophobic membrane barrier is of utmost interest as a pure basic and physicochemical question. In this Account, we focus on cationic cell-penetrating peptides (CPPs) and the way they cross cell membranes. We summarize the history of this field that emerged around 20 years ago. CPPs were indeed first identified as protein-transduction domains from the human immunodeficiency virus (HIV) TAT protein and the Antennapedia homeoprotein, a transcription factor from Drosophila. We highlight our contribution to the elucidation of CPP internalization pathways, in particular translocation, which implies perturbation and reorganization of the lipid bilayer, and endocytosis depending on sulfated glycosaminoglycans. We show a particular role of Trp (indole side chain) and Arg (guanidinium side chain), which are essential amino acids for CPP internalization. Interactions with the cell-surface are not only Coulombic; H-bonds and hydrophobic interactions contribute also significantly to CPP entry. The capacity of CPPs to cross cell membrane is not related to their strength of membrane binding. Finally, we present optimized methods based on mass spectrometry and fluorescence spectroscopy that allow unequivocal quantification of CPPs inside cells or bound to the outer leaflet of the membrane, and discuss some limitations of the technique of flow cytometry that we have recently highlighted.

Structure and Dynamics of an Intrinsically Disordered Protein Region That Partially Folds upon Binding by Chemical-Exchange NMR.

Many intrinsically disordered proteins (IDPs) and protein regions (IDRs) engage in transient, yet specific, interactions with a variety of protein partners. Often, if not always, interactions with a protein partner lead to partial folding of the IDR. Characterizing the conformational space of such complexes is challenging: in solution-state NMR, signals of the IDR in the interacting region become broad, weak, and often invisible, while X-ray crystallography only provides information on fully ordered regions. There is thus a need for a simple method to characterize both fully and partially ordered regions in the bound state of IDPs. Here, we introduce an approach based on monitoring chemical exchange by NMR to investigate the state of an IDR that folds upon binding through the observation of the free state of the protein. Structural constraints for the bound state are obtained from chemical shifts, and site-specific dynamics of the bound state are characterized by relaxation rates. The conformation of the interacting part of the IDR was determined and subsequently docked onto the structure of the folded partner. We apply the method to investigate the interaction between the disordered C-terminal region of Artemis and the DNA binding domain of Ligase IV. We show that we can accurately reproduce the structure of the core of the complex determined by X-ray crystallography and identify a broader interface. The method is widely applicable to the biophysical investigation of complexes of disordered proteins and folded proteins.

Antimicrobial activity of lysozyme isoforms: Key molecular features.

Increasing bacterial resistance towards antibiotics has stimulated research for novel antimicrobials. Proteins acting on bacterial membranes could be a solution. Lysozyme has been proven active against E. coli by disruption of both outer and cytoplasmic membranes, with dry-heating increasing lysozyme activity. Dry-heated lysozyme (DH-L) is a mixture of isoforms (isoaspartyl, native-like and succinimide lysozymes), giving rise to two questions: what effects does each form have, and which physicochemical properties are critical as regards the antibacterial activity? These issues were investigated by fractionating DH-L, analyzing structural properties of each fraction, and testing each fraction in vivo on bacteria and in vitro on membrane models. Positive net charge, hydrophobicity and molecular flexibility of the isoforms seem key parameters for their interaction with E. coli membranes. The succinimide lysozyme fraction, the most positive, flexible and hydrophobic, shows the highest antimicrobial activity, induces the strongest bacterial membrane disruption and is the most surface active on model lipid monolayers. Moreover, each fraction appears less efficient than DH-L against E. coli, indicating a synergetic cooperation between lysozyme isoforms. The bacterial membrane modifications induced by one isoform could facilitate the subsequent action of the other isoforms.

ECL1i, d(LGTFLKC), a novel, small peptide that specifically inhibits CCL2-dependent migration.

CC chemokine receptor type 2 (CCR2) is a key molecule in inflammatory diseases and is an obvious drug target for the treatment of inflammation. A number of nonpeptidic, competitive CCR2 antagonists have been developed, but none has yet been approved for clinical use. Our aim was to identify a short peptide that showed allosteric antagonism against human and mouse CCR2. On the basis of sequence analysis and 3-dimensional modeling, we identified an original 7-d-amino acid peptidic CCR2 inhibitor that we have called extracellular loop 1 inverso (ECL1i), d(LGTFLKC). In vitro, ECL1i selectively and potently inhibits CC chemokine ligand type 2 (CCL2)-triggered chemotaxis (IC50, 2 µM) but no other conventional CCL2-associated events. We used the classic competitive CCR2 antagonist, BMS22 {2-[(isopropylaminocarbonyl)amino]-N-[2-[[cis-2-[[4-(methylthio)benzoyl]amino]cyclohexyl]amino]-2-oxoethyl]-5-(trifluoromethyl)benzamide}, as positive control and inhibited CCL2-dependent chemotaxis with an IC50 of 18 nM. As negative control, we used a peptide with the same composition as ECL1i, but in a different sequence, d(FKLTLCG). In vivo, ECL1i (4 mg/kg) interfered with CCR2-positive cell recruitment and attenuated disease progression in experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. This study establishes ECL1i as the first allosteric inhibitor of CCR2 with functional selectivity. ECL1i is a promising new agent in therapeutic development, and it may, by its selective effect, increase our understanding of CCR2 signaling pathways and functions.-Auvynet, C., Baudesson de Chanville, C., Hermand, P., Dorgham, K., Piesse, C., Pouchy, C., Carlier, L., Poupel, L., Barthélémy, S., Felouzis, V., Lacombe, C., Sagan, S., Salomon, B., Deterre, P., Sennlaub, F., Combadière, C. ECL1i, d(LGTFLKC), a novel, small peptide that specifically inhibits CCL2-dependent migration.

A direct label-free MALDI-TOF mass spectrometry based assay for the characterization of inhibitors of protein lysine methyltransferases.

Histone lysine methylation is associated with essential biological functions like transcription activation or repression, depending on the position and the degree of methylation. This post-translational modification is introduced by protein lysine methyltransferases (KMTs) which catalyze the transfer of one to three methyl groups from the methyl donor S-adenosyl-L-methionine (AdoMet) to the amino group on the side chain of lysines. The regulation of protein lysine methylation plays a primary role not only in the basic functioning of normal cells but also in various pathologies and KMT deregulation is associated with diseases including cancer. These enzymes are therefore attractive targets for the development of new antitumor agents, and there is still a need for direct methodology to screen, identify, and characterize KMT inhibitors. We report here a simple and robust in vitro assay to quantify the enzymatic methylation of KMT by MALDI-TOF mass spectrometry. Following this protocol, we can monitor the methylation events over time on a peptide substrate. We detect in the same spectrum the modified and unmodified substrates, and the ratios of both signals are used to quantify the amount of methylated substrate. We first demonstrated the validity of the assay by determining inhibition parameters of two known inhibitors of the KMT SET7/9 ((R)-PFI-2 and sinefungin). Next, based on structural comparison with these inhibitors, we selected 42 compounds from a chemical library. We applied the MALDI-TOF assay to screen their activity as inhibitors of the KMT SET7/9. This study allowed us to determine inhibition constants as well as kinetic parameters of a series of SET7/9 inhibitors and to initiate a structure activity discussion with this family of compounds. This assay is versatile and can be easily adapted to other KMT substrates and enzymes as well as automatized.

Exploiting Benzophenone Photoreactivity To Probe the Phospholipid Environment and Insertion Depth of the Cell-Penetrating Peptide Penetratin in Model Membranes.

Penetratin (RQIKIWFQNRRMKWKK) enters cells by different mechanisms, including membrane translocation, thus implying that the peptide interacts with the lipid bilayer. Penetratin also crosses the membrane of artificial vesicles, depending on their phospholipid content. To evaluate the phospholipid preference of penetratin, as the first step of translocation, we exploited the benzophenone triplet kinetics of hydrogen abstraction, which is slower for secondary than for allylic hydrogen atoms. By using multilamellar vesicles of varying phospholipid content, we identified and characterized the cross-linked products by MALDI-TOF mass spectrometry. Penetratin showed a preference for negatively charged (vs. zwitterionic) polar heads, and for unsaturated (vs. saturated) and short (vs. long) saturated phospholipids. Our study highlights the potential of using benzophenone to probe the environment and insertion depth of membranotropic peptides in membranes.

The role of tryptophans on the cellular uptake and membrane interaction of arginine-rich cell penetrating peptides.

Cell-penetrating peptides (CPP) are able to efficiently transport cargos across cell membranes without being cytotoxic to cells, thus present a great potential in drug delivery and diagnosis. While the role of cationic residues in CPPs has been well studied, that of Trp is still not clear. Herein 7 peptide analogs of RW9 (RRWWRRWRR, an efficient CPP) were synthesized in which Trp were systematically replaced by Phe residues. Quantification of cellular uptake reveals that substitution of Trp by Phe strongly reduces the internalization of all peptides despite the fact that they strongly accumulate in the cell membrane. Cellular internalization and biophysical studies show that not only the number of Trp residues but also their positioning in the helix and the size of the hydrophobic face they form are important for their internalization efficacy, the highest uptake occurring for the analog with 3 Trp residues. Using CD and ATR-FTIR spectroscopy we observe that all peptides became structured in contact with lipids, mainly in α-helix. Intrinsic tryptophan fluorescence studies indicate that all peptides partition in the membrane in about the same manner (Kp~10(5)) and that they are located just below the lipid headgroups (~10 Å) with slightly different insertion depths for the different analogs. Plasmon Waveguide Resonance studies reveal a direct correlation between the number of Trp residues and the reversibility of the interaction following membrane washing. Thus a more interfacial location of the CPP renders the interaction with the membrane more adjustable and transitory enhancing its internalization ability.

Massive glycosaminoglycan-dependent entry of Trp-containing cell-penetrating peptides induced by exogenous sphingomyelinase or cholesterol depletion.

Among non-invasive cell delivery strategies, cell-penetrating peptide (CPP) vectors represent interesting new tools. To get fundamental knowledge about the still debated internalisation mechanisms of these peptides, we modified the membrane content of cells, typically by hydrolysis of sphingomyelin or depletion of cholesterol from the membrane outer leaflet. We quantified and visualised the effect of these viable cell surface treatments on the internalisation efficiency of different CPPs, among which the most studied Tat, R9, penetratin and analogues, that all carry the N-terminal biotin-Gly4 tag cargo. Under these cell membrane treatments, only penetratin and R6W3 underwent a massive glycosaminoglycan (GAG)-dependent entry in cells. Internalisation of the other peptides was only slightly increased, similarly in the absence or the presence of GAGs for R9, and only in the presence of GAGs for Tat and R6L3. Ceramide formation (or cholesterol depletion) is known to lead to the reorganisation of membrane lipid domains into larger platforms, which can serve as a trap and cluster receptors. These results show that GAG clustering, enhanced by formation of ceramide, is efficiently exploited by penetratin and R6W3, which contains Trp residues in their sequence but not Tat, R9 and R6L3. Hence, these data shed new lights on the differences in the internalisation mechanism and pathway of these peptides that are widely used in delivery of cargo molecules.

A proapoptotic peptide conjugated to penetratin selectively inhibits tumor cell growth.

The peptide KLA (acetyl-(KLAKLAK)2-NH2), which is rather non toxic for eukaryotic cell lines, becomes active when coupled to the cell penetrating peptide, penetratin (Pen), by a disulfide bridge. Remarkably, the conjugate KLA-Pen is cytotoxic, at low micromolar concentrations, against a panel of seven human tumor cell lines of various tissue origins, including cells resistant to conventional chemotherapy agents but not to normal human cell lines. Live microscopy on cells possessing fluorescent labeled mitochondria shows that in tumor cells, KLA-Pen had a strong impact on mitochondria tubular organization instantly resulting in their aggregation, while the unconjugated KLA and pen peptides had no effect. But, mitochondria in various normal cells were not affected by KLA-Pen. The interaction with membrane models of KLA-Pen, KLA and penetratin were studied using dynamic light scattering, calorimetry, plasmon resonance, circular dichroism and ATR-FTIR to unveil the mode of action of the conjugate. To understand the selectivity of the conjugate towards tumor cell lines and its action on mitochondria, lipid model systems composed of zwitterionic lipids were used as mimics of normal cell membranes and anionic lipids as mimics of tumor cell and mitochondria membrane. A very distinct mode of interaction with the two model systems was observed. KLA-Pen may exert its deleterious and selective action on cancer cells by the formation of pores with an oblique membrane orientation and establishment of important hydrophobic interactions. These results suggest that KLA-Pen could be a lead compound for the design of cancer therapeutics.

Label-free measurement of histone lysine methyltransferases activity by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Histone lysine methyltransferases (HKMTs) are enzymes that play an essential role in epigenetic regulation. Thus, identification of inhibitors specifically targeting these enzymes represents a challenge for the development of new antitumor therapeutics. Several methods for measuring HKMT activity are already available. Most of them use indirect measurement of the enzymatic reaction through radioactive labeling or antibody-recognized products or coupled enzymatic assays. Mass spectrometry (MS) represents an interesting alternative approach because it allows direct detection and quantification of enzymatic reactions and can be used to determine kinetics and to screen small molecules as potential inhibitors. Application of mass spectrometry to the study of HKMTs has not been fully explored yet. We describe here the development of a simple reliable label-free MALDI-TOF MS-based assay for the detection and quantification of peptide methylation, using SET7/9 as a model enzyme. Importantly, the use of expensive internal standard often required in mass spectrometry quantitative analysis is not necessary in this assay. This MS assay allowed us to determine enzyme kinetic parameters as well as IC50 for a known inhibitor of this enzyme. Furthermore, a comparative study with an antibody-based immunosorbent assay showed that the MS assay is more reliable and suitable for the screening of inhibitors.

Tryptophan within basic peptide sequences triggers glycosaminoglycan-dependent endocytosis.

Deciphering the structural requirements and mechanisms for internalization of cell-penetrating peptides (CPPs) is required to improve their delivery efficiency. Herein, a unique role of tryptophan (Trp) residues in the interaction and structuring of cationic CPP sequences with glycosaminoglycans (GAGs) has been characterized, in relation with cell internalization. Using isothermal titration calorimetry, circular dichroism, NMR, mass spectrometry, and phase-contrast microscopy, we compared the interaction of 7 basic CPPs with 5 classes of GAGs. We found that the affinity of CPPs for GAGs increases linearly with the number of Trp residues, from 30 nM for a penetratin analog with 1 Trp residue to 1.5 nM for a penetratin analog with 6 Trp residues for heparin (HI); peptides with Trp residues adopt a predominantly ß-strand structure in complex with HI and form large, stable ß-sheet aggregates with GAGs; and in the absence of any cytotoxicity effect, the quantity of peptide internalized into CHO cells increased 2 times with 1 Trp residue, 10 times with 2 Trp residues, and 20 times with 3 Trp residues, compared with +6 peptides with no Trp residues. Therefore, Trp residues represent molecular determinants in basic peptide sequences not only for direct membrane translocation but also for efficient endocytosis through GAGs.

Membrane interactions of two arginine-rich peptides with different cell internalization capacities.

Cell penetrating peptides (CPPs) can cross cell membranes in a receptor independent manner and transport cargo molecules inside cells. These peptides can internalize through two independent routes: energy dependent endocytosis and energy independent translocation across the membrane, but the exact mechanisms are still unknown. The interaction of the CPP with different membrane components is certainly a preliminary key point that triggers internalization, such as the interaction with lipids to lead to the translocation process. In this study, we used two arginine-rich peptides, RW9 (RRWWRRWRR-NH2), which is a potent CPP, and RL9 (RRLLRRLRR-NH2) that, although binding tightly and accumulating on membranes, does not enter into cells. Using a set of experimental and theoretical techniques, we studied the binding, insertion and orientation of the peptides into different model membranes as well as the subsequent membrane reorganization. Herein we show that although the two peptides had rather similar behavior regarding lipid membrane interaction, subtle differences were found concerning the depth of peptide insertion, effect on the lipid chain ordering and kinetics of peptide insertion in the membrane, which altogether might explain their different cell internalization capacities. Molecular dynamics simulation studies show that some peptide molecules flipped their orientation over the course of the simulation such that the hydrophobic residues penetrated deeper in the lipid core region while Arg-residues maintained H-bonds with the lipid headgroups, serving as a molecular hinge in a conformation that appeared to correspond to the equilibrium one.