DNA-Interactive Properties of Crotamine,a Cell-Penetrating Polypeptide and a Potential Drug Carrier

Crotamine, a 42-residue polypeptide derived from the venom of the South American rattlesnake Crotalus durissus terrificus, has been shown to be a cell-penetrating protein that targets chromosomes, carries plasmid DNA into cells, and shows specificity for actively proliferating cells. Given this potential role as a nucleic acid-delivery vector, we have studied in detail the binding of crotamine to single- and double-stranded DNAs of different lengths and base compositions over a range of ionic conditions. Agarose gel electrophoresis and ultraviolet spectrophotometry analysis indicate that complexes of crotamine with long-chain DNAs readily aggregate and precipitate at low ionic strength. This aggregation, which may be important for cellular uptake of DNA, becomes less likely with shorter chain length. 25-mer oligonucleotides do not show any evidence of such aggregation, permitting the determination of affinities and size via fluorescence quenching experiments. The polypeptide binds non-cooperatively to DNA, covering about 5 nucleotide residues when it binds to single (ss) or (ds) double stranded molecules. The affinities of the protein for ss- vs. ds-DNA are comparable, and inversely proportional to salt levels. Analysis of the dependence of affinity on [NaCl] indicates that there are a maximum of ∼3 ionic interactions between the protein and DNA, with some of the binding affinity attributable to non-ionic interactions. Inspection of the three-dimensional structure of the protein suggests that residues 31 to 35, Arg-Trp-Arg-Trp-Lys, could serve as a potential DNA-binding site. A hexapeptide containing this sequence displayed a lower DNA binding affinity and salt dependence as compared to the full-length protein, likely indicative of a more suitable 3D structure and the presence of accessory binding sites in the native crotamine. Taken together, the data presented here describing crotamine-DNA interactions may lend support to the design of more effective nucleic acid drug delivery vehicles which take advantage of crotamine as a carrier with specificity for actively proliferating cells.     

Molecular characterization of the interaction of crotamine-derived nucleolar targeting peptides with lipid membranes

A novel class of cell-penetrating, nucleolar-targeting peptides (NrTPs), was recently developed from the rattlesnake venom toxin crotamine. Based on the intrinsic fluorescence of tyrosine or tryptophan residues, the partition of NrTPs and crotamine to membranes with variable lipid compositions was studied. Partition coefficient values (in the 10(2)-10(5) range) followed essentially the compositional trend POPC:POPG≤POPG相似文献   

Properties of cell penetrating peptides (CPPs)

Different approaches have been developed for the introduction of macromolecules, proteins and DNA into target cells. Viral (retroviruses, lentiviruses, etc.) and nonviral (liposomes, bioballistics etc.) vectors as well as lipid particles have been tested as DNA delivery systems. However, all of them share several undesirable effects that are difficult to overcome, such as unwanted immunoresponse and limited cell targeting. The discovery of the cell penetrating peptides (CPPs) showing properties of macromolecules carriers and enhancers of viral vectors, opened new opportunities for the delivery of biologically active cargos, including therapeutically relevant genes into various cells and tissues. This review summarizes recent data about the best characterized CPPs as well as those sharing cell-penetrating and cargo delivery properties despite differing in the primary sequence. The putative mechanisms of CPPs penetration into cells and interaction with intracellular structures such as chromosomes, cytoskeleton and centrioles are addressed. We further discuss recent developments in overcoming the lack of cells specificity, one of the main obstacles for CPPs application in gene therapy. In particular, we review a newly discovered affinity of CPPs to actively proliferating cells.     

Arginine-rich cell-penetrating peptides deliver gene into living human cells

Transgenesis is a process that introduces exogenous nucleic acids into the genome of an organism to produce desired traits or evaluate function. Improvements of transgenic technologies are always important pursuit in the last decades. Recently, cell-penetrating peptides (CPPs) were studied as shuttles that can internalize into cells directly and serve as carriers to deliver different cargoes into cells. In the present study, we evaluate whether arginine-rich CPPs can be used for gene delivery into human cells in a noncovalent fashion. We demonstrate that three arginine-rich CPPs (SR9, HR9, and PR9) are able to transport plasmid DNA into human A549 cells. For the functional gene assay, the CPP-delivered plasmid DNA containing the enhanced green fluorescent protein (EGFP) coding sequence could be actively expressed in cells. The treatment of calcium chloride did not facilitate the CPP-mediated transfection efficiency, but enhance the gene expression intensity. Mechanistic studies further revealed that HR9/DNA complexes mediate the direct membrane translocation pathway for gene delivery. Our results suggest that arginine-rich CPPs, especially HR9, appear to be a high efficient and promising tool for gene transfer.     

Efficient penetration of Scp01-b and its DNA transfer abilities into cells

The in vivo application potential of viral-based gene delivery approaches is hindered by a risk of insertional oncogenesis. Of the many delivery methods, cell-penetrating peptides (CPP)-based delivery has good biocompatibility and biodegradability. However, low efficiency is still the disadvantage of CPPs-based nucleic acid transfection, and delivery efficiency may vary from different CPPs. Here, we describe Scp01-b, as a new CPP, which can enter cultured cell lines and primary cultured cells examined by fluorescence microscopy and quantitative assay, the internalization process is a concentration, temperature, and incubation time-dependent manner. Scp01-b does not insert into the membrane directly and its uptake is mediated through endocytosis pathway. Moreover, Scp01-b could mediate the uptake of plasmid DNA into the Caski and HSC-T6 cells, and we noted that Scp01-b-mediated transfection efficiency was nearly the same with traditional liposome (TurboFectin)-mediated transfection. These findings suggest that Scp01-b can act as a useful tool for non-viral-based delivery in further application such as reprogramming and gene editing.     

New generation CPPs show distinct selectivity for cancer and noncancer cells

In the last three decades, many new cell-penetrating peptides (CPPs) were developed that exhibited enhanced cell selectivity. Thus, we aimed to validate the tumor cell selectivity of peptides from this new generation, namely fragments mini-crotamine and mini-maurocalcine. Both of these peptides are derived from venoms. Furthermore, we studied an analog of the classical CPP HIV-TAT(47-57) with alternating chirality of Arg residues. To allow covalent coupling of cargoes or fluorophores, a cysteine residue was introduced to the N-terminus of the synthesized peptides. The therapeutic antibody trastuzumab conjugated to different fluorescent dyes was used for internalization studies. Comparison of uptake efficiencies revealed that CPPs of the new generation are in contrast to MPG-peptides, nearly unable to internalize the noncovalently formed complexes with trastuzumab. Interestingly, the fluorescent derivative of the crotamine fragment was mainly observed in a subpopulation of breast cancer cells, whereas it was homogenously distributed in fibrosarcoma, colon cancer, and noncancerous endothelia cells. Thus, the fluorescent crotamine fragment reported herein is a potent theranostic tool for image-guided applications. This peptide can be used to pinpoint the level of heterogeneity present within tumors and aid in the generation of therapeutics that target heterogenic subpopulations.     

Gene Delivery to Tobacco Root Cells with Single-Walled Carbon Nanotubes and Cell-Penetrating Fusogenic Peptides

Development of efficient, easy, and safe gene delivery methods is of great interest in the field of plant biotechnology. Considering the limitations of the usual transfection methods (such as transgene size and plant type), several new techniques have been tested for replacement. The success of some biological and synthetic nanostructures such as cell-penetrating peptides and carbon nanotubes in transferring macromolecules (proteins and nucleic acids) into mammalian cells provoked us to assess the ability of an engineered chimeric peptide and also arginine functionalized single-walled carbon nanotube in gene delivery to intact tobacco (Nicotiana tabacum var. Virginia) root cells. It was suggested that the engineered peptide with its special cationic and hydrophobic domains and the arginine functionalized single-walled carbon nanotube due to its nano-cylindrical shape can pass plant cell barriers while plasmid DNA (which codes green fluorescent protein) has been condensed on them. The success of gene delivery to tobacco root cells was confirmed by fluorescence microscopy and western blotting analysis.     

Cell-penetrating and cell-targeting peptides in drug delivery

During the last decade, the potential of peptides for drug delivery into cells has been highlighted by the discovery of several cell-penetrating peptides (CPPs). CPPs are very efficient in delivering various molecules into cells. However, except in some specific cases, their lack of cell specificity remains the major drawback for their clinical development. At the same time, various peptides with specific binding activity for a given cell line (cell-targeting peptides) have also been reported in the literature. One of the goals of the next years will be to optimize the tissue and cell delivery of therapeutic molecules by means of peptides which combine both targeting and internalization advantages. In this review, we describe the main strategies that are currently in use or likely to be employed in the near future to associate both targeting and delivery properties.     

Hydrophobic and electrostatic interactions between cell penetrating peptides and plasmid DNA are important for stable non‐covalent complexation and intracellular delivery

Cell penetrating peptides are useful tools for intracellular delivery of nucleic acids. Delivery of plasmid DNA, a large nucleic acid, poses a challenge for peptide mediated transport. The paper investigates and compares efficacy of five novel peptide designs for complexation of plasmid DNA and subsequent delivery into cells. The peptides were designed to contain reported DNA condensing agents and basic cell penetrating sequences, octa‐arginine (R₈) and CHK₆HC coupled to cell penetration accelerating peptides such as Bax inhibitory mutant peptide (KLPVM) and a peptide derived from the Kaposi fibroblast growth factor (kFGF) membrane translocating sequence. A tryptophan rich peptide, an analogue of Pep‐3, flanked with CH₃ on either ends was also a part of the study. The peptides were analysed for plasmid DNA complexation, protection of peptide–plasmid DNA complexes against DNase I, serum components and competitive ligands by simple agarose gel electrophoresis techniques. Hemolysis of rat red blood corpuscles (RBCs) in the presence of the peptides was used as a measure of peptide cytotoxicity. Plasmid DNA delivery through the designed peptides was evaluated in two cell lines, human cervical cancer cell line (HeLa) and (NIH/3 T3) mouse embryonic fibroblasts via expression of the secreted alkaline phosphatase (SEAP) reporter gene. The importance of hydrophobic sequences in addition to cationic sequences in peptides for non‐covalent plasmid DNA complexation and delivery has been illustrated. An alternative to the employment of fatty acid moieties for enhanced gene transfer has been proposed. Comparison of peptides for plasmid DNA complexation and delivery of peptide–plasmid DNA complexes to cells estimated by expression of a reporter gene, SEAP. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Efficient gene transfer by histidylated polylysine/pDNA complexes.

Plasmid/polylysine complexes, which are used to transfect mammalian cells, increase the uptake of DNA, but plasmid molecules are sequestered into vesicles where they cannot escape to reach the nuclear machinery. However, the transfection efficiency increases when membrane-disrupting reagents such as chloroquine or fusogenic peptides, are used to disrupt endosomal membranes and to favor the delivery of plasmid into the cytosol. We designed a cationic polymer that forms complexes with a plasmid DNA (pDNA) and mediates the transfection of various cell lines in the absence of chloroquine or fusogenic peptides. This polymer is a polylysine (average degree of polymerization of 190) partially substituted with histidyl residues which become cationic upon protonation of the imidazole groups at pH below 6.0. The transfection efficiency was optimal with a polylysine having 38 +/- 5% of the epsilon-amino groups substituted with histidyl residues; it was not significantly impaired in the presence of serum in the culture medium. The transfection was drastically inhibited in the presence of bafilomycin A1, indicating that the protonation of the imidazole groups in the endosome lumen might favor the delivery of pDNA into the cytosol.     

Cell-penetrating peptides: breaking through to the other side

Cell-penetrating peptides (CPPs) have been previously shown to be powerful transport vector tools for the intracellular delivery of a large variety of cargoes through the cell membrane. Intracellular delivery of plasmid DNA (pDNA), oligonucleotides, small interfering RNAs (siRNAs), proteins and peptides, contrast agents, drugs, as well as various nanoparticulate pharmaceutical carriers (e.g., liposomes, micelles) has been demonstrated both in vitro and in vivo. This review focuses on the peptide-based strategy for intracellular delivery of CPP-modified nanocarriers to deliver small molecule drugs or DNA. In addition, we discuss the rationales for the design of 'smart' pharmaceutical nanocarriers in which the cell-penetrating properties are hidden until triggered by exposure to appropriate environmental conditions (e.g., a particular pH, temperature, or enzyme level), applied local microwave, ultrasound, or radiofrequency radiation.     

Inhibition of malaria parasite Plasmodium falciparum development by crotamine,a cell penetrating peptide from the snake venom

We show here that crotamine, a polypeptide from the South American rattlesnake venom with cell penetrating and selective anti-fungal and anti-tumoral properties, presents a potent anti-plasmodial activity in culture. Crotamine inhibits the development of the Plasmodium falciparum parasites in a dose-dependent manner [IC₅₀ value of 1.87 μM], and confocal microscopy analysis showed a selective internalization of fluorescent-labeled crotamine into P. falciparum infected erythrocytes, with no detectable fluorescence in uninfected healthy erythrocytes. In addition, similarly to the crotamine cytotoxic effects, the mechanism underlying the anti-plasmodial activity may involve the disruption of parasite acidic compartments H⁺ homeostasis. In fact, crotamine promoted a reduction of parasites organelle fluorescence loaded with the lysosomotropic fluorochrome acridine orange, in the same way as previously observed mammalian tumoral cells. Taken together, we show for the first time crotamine not only compromised the metabolism of the P. falciparum, but this toxin also inhibited the parasite growth. Therefore, we suggest this snake polypeptide as a promising lead molecule for the development of potential new molecules, namely peptidomimetics, with selectivity for infected erythrocytes and ability to inhibit the malaria infection by its natural affinity for acid vesicles.     

Gene transport and expression by arginine-rich cell-penetrating peptides in Paramecium

Owing to the cell membrane barriers, most macromolecules and hydrophilic molecules could not freely enter into living cells. However, cell-penetrating peptides (CPPs) have been discovered that can translocate themselves and associate cargoes into the cytoplasm. In this study, we demonstrate that three arginine-rich CPPs (SR9, HR9 and PR9) can form stable complexes with plasmid DNA at the optimized nitrogen/phosphate ratio of 3 and deliver plasmid DNA into Paramecium caudatum in a noncovalent manner. Accordingly, the transported plasmid encoding the green fluorescent protein (GFP) gene could be expressed in cells functionally assayed at both the protein and DNA levels. The efficiency of gene delivery varied among these CPPs in the order of HR9 > PR9 > SR9. In addition, these CPPs and CPP/DNA complexes were not cytotoxic in Paramecium detected by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diohenyltetrazolium bromide (MTT) assay. Thus, these results suggest that the functionality of arginine-rich CPPs offers an efficient and safe tool for transgenesis in eukaryotic protozoans.     

Antibacterial activity and dual mechanisms of peptide analog derived from cell-penetrating peptide against Salmonella typhimurium and Streptococcus pyogenes

A number of research have proven that antimicrobial peptides are of greatest potential as a new class of antibiotics. Antimicrobial peptides and cell-penetrating peptides share some similar structure characteristics. In our study, a new peptide analog, APP (GLARALTRLLRQLTRQLTRA) from the cell-penetrating peptide ppTG20 (GLFRALLRLLRSLWRLLLRA), was identified simultaneously with the antibacterial mechanism of APP against Salmonella typhimurium and Streptococcus pyogenes. APP displayed potent antibacterial activity against Gram-negative and Gram-positive strains. The minimum inhibitory concentration was in the range of 2 to 4 μM. APP displayed higher cell selectivity (about 42-fold increase) as compared to the parent peptide for it decreased hemolytic activity and increased antimicrobial activity. The calcein leakage from egg yolk l-α-phosphatidylcholine (EYPC)/egg yolk l-α-phosphatidyl-dl-glycerol and EYPC/cholesterol vesicles demonstrated that APP exhibited high selectivity. The antibacterial mechanism analysis indicated that APP induced membrane permeabilization in a kinetic manner for membrane lesions allowing O-nitrophenyl-β-d-galactoside uptake into cells and potassium release from APP-treated cells. Flow cytometry analysis demonstrated that APP induced bacterial live cell membrane damage. Circular dichroism, fluorescence spectra, and gel retardation analysis confirmed that APP interacted with DNA and intercalated into the DNA base pairs after penetrating the cell membrane. Cell cycle assay showed that APP affected DNA synthesis in the cell. Our results suggested that peptides derived from the cell-penetrating peptide have the potential for antimicrobial agent development, and APP exerts its antibacterial activity by damaging bacterial cell membranes and binding to bacterial DNA to inhibit cellular functions, ultimately leading to cell death.     

Vesicle membrane interactions of penetratin analogues

Reports on serious artifacts associated with the use of cell fixation in studies of the cellular uptake of cell-penetrating peptides, also denoted protein transduction domains, have demonstrated the need for a reevaluation of the current understanding of peptide-mediated cellular delivery of large, hydrophilic molecules. In a recent study on the internalization in unfixed cells of penetratin and its analogues in which tryptophans are substituted for phenylalanines (Pen2W2F), lysines for arginines (PenArg), and arginines for lysines (PenLys), we revealed large dissimilarities in cell interactions among the peptides [Thorén et al. (2003) Biochem. Biophys. Res. Commun. 307, 100-107]. We here investigated possible correlations with their respective affinities for the lipid membranes of large unilamellar vesicles. The variations found in membrane affinity correlated qualitatively with differences in hydrophobicity among the peptides but were by far too small to account for the striking differences in cell membrane binding. Interestingly, we found that the inclusion of a small fraction of lipids conjugated to poly(ethylene glycol) (PEG) in the vesicles both stabilized the vesicle dispersion against peptide-induced aggregation and, furthermore, enhanced the binding of the peptides to the membrane. By use of PEG-conjugated lipids, it could be shown that vesicle aggregation drives an alpha-helix to beta-sheet conformational transition for these peptides. A similar transition was discovered at submicellar concentrations of sodium dodecyl sulfate in aqueous solution for all peptides except PenLys. Finally, significant changes of the contributions to CD spectra from aromatic residues due to their insertion into the membrane were observed.     

Translocation of dynorphin neuropeptides across the plasma membrane. A putative mechanism of signal transmission

Several peptides, including penetratin and Tat, are known to translocate across the plasma membrane. Dynorphin opioid peptides are similar to cell-penetrating peptides in a high content of basic and hydrophobic amino acid residues. We demonstrate that dynorphin A and big dynorphin, consisting of dynorphins A and B, can penetrate into neurons and non-neuronal cells using confocal fluorescence microscopy/immunolabeling. The peptide distribution was characterized by cytoplasmic labeling with minimal signal in the cell nucleus and on the plasma membrane. Translocated peptides were associated with the endoplasmic reticulum but not with the Golgi apparatus or clathrin-coated endocytotic vesicles. Rapid entry of dynorphin A into the cytoplasm of live cells was revealed by fluorescence correlation spectroscopy. The translocation potential of dynorphin A was comparable with that of transportan-10, a prototypical cell-penetrating peptide. A central big dynorphin fragment, which retains all basic amino acids, and dynorphin B did not enter the cells. The latter two peptides interacted with negatively charged phospholipid vesicles similarly to big dynorphin and dynorphin A, suggesting that interactions of these peptides with phospholipids in the plasma membrane are not impaired. Translocation was not mediated via opioid receptors. The potential of dynorphins to penetrate into cells correlates with their ability to induce non-opioid effects in animals. Translocation across the plasma membrane may represent a previously unknown mechanism by which dynorphins can signal information to the cell interior.     

The human antimicrobial peptide LL-37 transfers extracellular DNA plasmid to the nuclear compartment of mammalian cells via lipid rafts and proteoglycan-dependent endocytosis

Antimicrobial peptides, such as LL-37, are found both in nonvertebrates and vertebrates, where they represent important components of innate immunity. Bacterial infections at epithelial surfaces are associated with substantial induction of LL-37 expression, which allows efficient lysis of the invading microbes. Peptide-mediated lysis results in the release of bacterial nucleic acids with potential pathobiological activity in the host. Here, we demonstrate that LL-37 targets extracellular DNA plasmid to the nuclear compartment of mammalian cells, where it is expressed. DNA transfer occurred at physiological LL-37 concentrations that killed bacterial cells, whereas virtually no cytotoxic or growth-inhibitory effects were observed in mammalian cells. Furthermore, LL-37 protected DNA from serum nuclease degradation. LL-37.DNA complex uptake was a saturable time- and temperature-dependent process and was sensitive to cholesterol-depleting agents that are known to disrupt lipid rafts and caveolae, as shown by flow cytometry. Confocal fluorescence microscopy studies showed localization of internalized DNA to compartments stained by cholera toxin B, a marker of lipid rafts, but failed to demonstrate any co-localization of internalized DNA with caveolin-positive endocytotic vesicles. Moreover, LL-37-mediated plasmid uptake and reporter gene expression were strictly dependent on cell surface proteoglycans. We conclude that the human antimicrobial peptide LL-37 binds to, protects, and efficiently targets DNA plasmid to the nuclei of mammalian cells through caveolae-independent membrane raft endocytosis and cell surface proteoglycans.     

A novel amphipathic cell-penetrating peptide based on the N-terminal glycosaminoglycan binding region of human apolipoprotein E

In the direct cell membrane penetration, arginine-rich cell-penetrating peptides are thought to penetrate into cells across the hydrophobic lipid membranes. To investigate the effect of the amphipathic property of arginine-rich peptide on the cell-penetrating ability, we designed a novel amphipathic cell-penetrating peptide, A2-17, and its derivative, A2-17KR, in which all lysine residues are substituted with arginine residues, based on the glycosaminoglycan binding region in the N-terminal α-helix bundle of human apolipoprotein E. Isothermal titration calorimetry showed that A2-17 variants have a strong ability to bind to heparin with high affinity. Circular dichroism and tryptophan fluorescence measurements demonstrated that A2-17 variants bind to lipid vesicles with a structural change from random coil to amphipathic α-helix, being inserted into the hydrophobic membrane interiors. Flow cytometric analysis and confocal laser scanning microscopy demonstrated the great cell penetration efficiency of A2-17 variants into CHO-K1 cells when incubated at low peptide concentrations (2 μM or less), suggesting that the increased amphipathicity with α-helix formation enhances the cell membrane penetration ability of arginine-rich peptides. Interestingly, A2-17KR exhibited lower efficiency of cell membrane penetration compared to A2-17 despite of their similar binding affinity to lipid membranes. Since high peptide concentrations (typically >10 μM) are usually prerequisite for efficient cell penetration of arginine-rich peptides, A2-17 is a unique amphipathic cell-penetrating peptide that exhibits an efficient cell penetration ability even at low peptide concentrations.     

Kinetic uptake profiles of cell penetrating peptides in lymphocytes and monocytes

Background

Nucleolar targeting peptides (NrTPs), resulting from structural minimization of the rattlesnake toxin crotamine, are a novel family of cell-penetrating peptides (CPPs) shown to internalize and deliver cargos into different cell types.

Methods

In this study, we address NrTP kinetics of translocation into primary cells. We used flow cytometry to measure the intracellular uptake of rhodamine B-labeled NrTPs in peripheral blood mononucleated cells (PBMCs).

Results

The kinetic profiles for each peptide are concentration-independent but significantly different among NrTPs, pointing out for the amino acid sequence importance. Arginine-containing peptides (NrTP7 and Tat_48–60, used for comparison) were found to be more toxic than lysine-containing ones, as expected. On the other hand, one same peptide behaves differently in each of the lymphocyte and monocyte cell populations, suggesting differences in entry mechanism that in turn reflect diversity in cell functionality. Uptake results obtained at 4 °C or using chemical endocytosis inhibitors support the importance of non-endocytic mechanisms in the cellular internalization of NrTP1 and NrTP5, while confirming endocytosis as the main mechanism of NrTPs entry.

Conclusion

Overall, both direct translocation and endocytosis mechanisms play a role in NrTP entry. Yet, there is predominance of endocytosis-mediated mechanisms. NrTPs (especially NrTP6) are an excellent intracellular delivery tool, with efficient internalization and no toxicity.

General significance

This work validates NrTPs as potential therapeutic tools for, e.g., cancer or inhibition of viral replication and establishes a new comparative and quantitative method to test CPP efficiency.     

Measuring peptide translocation into large unilamellar vesicles

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors(1). Many of these are referred to as cell-penetrating peptides, which are frequently noted for their potential as drug delivery vectors(1-3). Moreover, there is increasing interest in antimicrobial peptides that operate via non-membrane lytic mechanisms(4,5), particularly those that cross bacterial membranes without causing cell lysis and kill cells by interfering with intracellular processes(6,7). In fact, authors have increasingly pointed out the relationship between cell-penetrating and antimicrobial peptides(1,8). A firm understanding of the process of membrane translocation and the relationship between peptide structure and its ability to translocate requires effective, reproducible assays for translocation. Several groups have proposed methods to measure translocation into large unilamellar lipid vesicles (LUVs)(9-13). LUVs serve as useful models for bacterial and eukaryotic cell membranes and are frequently used in peptide fluorescent studies(14,15). Here, we describe our application of the method first developed by Matsuzaki and co-workers to consider antimicrobial peptides, such as magainin and buforin II(16,17). In addition to providing our protocol for this method, we also present a straightforward approach to data analysis that quantifies translocation ability using this assay. The advantages of this translocation assay compared to others are that it has the potential to provide information about the rate of membrane translocation and does not require the addition of a fluorescent label, which can alter peptide properties(18), to tryptophan-containing peptides. Briefly, translocation ability into lipid vesicles is measured as a function of the Foster Resonance Energy Transfer (FRET) between native tryptophan residues and dansyl phosphatidylethanolamine when proteins are associated with the external LUV membrane (Figure 1). Cell-penetrating peptides are cleaved as they encounter uninhibited trypsin encapsulated with the LUVs, leading to disassociation from the LUV membrane and a drop in FRET signal. The drop in FRET signal observed for a translocating peptide is significantly greater than that observed for the same peptide when the LUVs contain both trypsin and trypsin inhibitor, or when a peptide that does not spontaneously cross lipid membranes is exposed to trypsin-containing LUVs. This change in fluorescence provides a direct quantification of peptide translocation over time.