Liposomal cargo unloading induced by pH-sensitive peptides.

Amphiphilic peptides designed to have a pH-dependent conformational change and membrane activity are described. At physiologic pH, the peptides would exist in a random coil conformation, but at endosomal pH values they would switch to amphiphilic alpha-helices, disrupt membranes, and release liposomal contents. A series of peptides have been investigated that contain a high percentage of Glu residues for the pH-induced conformational switch, and Leu residues for optimal lipid binding. Circular dichroism (CD) results in aqueous and liposomal environments were performed and demonstrate a pH-dependent shift to helicity upon acidification. Liposomal release data at neutral and acidic pH, also document the success of this design strategy.     

Self-promoted cellular uptake of peptide/DNA transfection complexes

The designed alpha-helical amphipathic peptide LAH4 assembles several properties, which makes it an interesting candidate as a gene-delivery vehicle. Besides being short and soluble in aqueous solutions, LAH4 presents cationic residues, which allow for efficient complexation of DNA. In addition, this peptide is poorly hemolytic at neutral pH, while it is able to destabilize biological membranes in acidic conditions. In this study, the structure of the peptide/DNA transfection complex was examined by circular dichroism and solid-state nuclear magnetic resonance spectroscopies and the thermodynamics of its formation and disassembly was monitored in a quantitative manner as a function of pH by isothermal titration calorimetry. Notably, the number of peptides within the complex considerably decreases upon acidification of the medium. This observation has direct and important consequences for the mechanism of action because the acidification of the endosome results in high local concentrations of free peptide in this organelle. Thus, these peptides become available to interact with the endosomal membranes and thereby responsible for the delivery of the transfection complex to the cytoplasm. When these data are taken together, they indicate a dual role of the peptide during the transfection process, namely, DNA complexation and membrane permeabilization.     

Functional characterization of an endosome-disruptive peptide and its application in cytosolic delivery of immunoliposome-entrapped proteins

Antibody-directed liposomes (immunoliposomes) are frequently used for targeted drug delivery. However, delivery of large biotherapeutic molecules (i.e. peptides, proteins, or nucleic acids) with immunoliposomes is often hampered by an inefficient cytosolic release of entrapped macromolecules after target cell binding and subsequent endocytosis of immunoliposomes. To enhance cytosolic drug delivery from immunoliposomes present inside endosomes, a pH-dependent fusogenic peptide (diINF-7) resembling the NH(2)-terminal domain of influenza virus hemagglutinin HA-2 subunit was used. Functional characterization of this dimeric peptide showed its ability to induce fusion between liposome membranes and leakage of liposome-entrapped compounds when exposed to low pH. In a second series of experiments, diINF-7 peptides were encapsulated in immunoliposomes to enhance the endosomal escape of diphtheria toxin A chain (DTA), which inhibits protein synthesis when delivered into the cytosol of target cells. Immunoliposomes targeted to the internalizing epidermal growth factor receptor on the surface of ovarian carcinoma cells (OVCAR-3) and containing encapsulated DTA did not show any cytotoxicity toward OVCAR-3 cells. Cytotoxicity was only observed when diINF-7 peptides and DTA were co-encapsulated in the immunoliposomes. Thus, diINF-7 peptides entrapped inside liposomes can greatly enhance cytosolic delivery of liposomal macromolecules by pH-dependent destabilization of endosomal membranes after cellular uptake of liposomes.     

Adsorption equilibrium in immunoaffnity chromatography with antibodies to synthetic peptides

The effects of charged residues in peptide antigens on the binding characteristics of polyclonal antipeptide antibodies were studied using immunoadsorbents prepared by coupling the antibodies to CNBr-activated Sepharose 4B. Among the antipeptide antibodies, an antibody to the peptide without charged residues showed the most stable interaction with the peptide to the changes in pH. Conversely, the binding affinity of antibodies to the pep-tides with histidine residues having a unique pKa value of 6.0 decreased steeply with pH at around 6.0. The binding affinity of an antibody to the peptide with many charged residues decreased steeply with an increase in the ionic strength (adjusted by NaCl). Since circular dichroism (CD) spectrum measurements indicate that these peptides show disordered structures in the pH range of adsorption measurement, the dependence of peptide-antibody interaction on environmental conditions is attributed to the characteristics of side chains of the peptides. These results indicate that the dependence of the binding affinity of antipeptide antibodies on pH and the ionic strength is dominantly affected by the number and the pKa values of charged residues in the peptides.     

An alpha-helical peptide model for electrostatic interactions of proteins with DNA. The N terminus of RecA

A series of synthetic peptides have been studied as models for non-specific protein-DNA interactions. In an alpha-helical conformation, the charged amino acid residues of the N-terminal 24 residues of RecA protein are asymmetrically distributed; at neutral pH there is a +4 charge on one face of the helix and a -3 charge on the other face. Modeling suggests that the positive face of the helix can bind five DNA phosphate groups by electrostatic interactions. Circular dichroism (c.d.) spectra indicate that the analogous peptide, Rec24 (AIDENKQKALAAALGQIEKQFGKG-amide), is largely unstructured in water but becomes highly helical in the presence of DNA. Peptide titrations of fluorescent etheno-DNA confirm that the changes in the c.d. spectrum of the peptide are associated with binding, although a dependence of the c.d. signal on the degree of DNA saturation is observed, indicating that peptide can be bound in more than one conformation. At saturation the peptide binds to 5.0(+/- 0.5) DNA phosphate groups as predicted and the electrostatic nature of the binding is confirmed by a strong dependence on salt concentration. A "mutant" peptide where an acidic glutamate residue replaces an alanine on the basic face of the Rec24 helix exhibits weaker binding to single-stranded DNA, also consistent with the electrostatic nature of the proposed peptide-DNA interaction. Extending Rec24 by ten amino acid residues, where the additional residues do not participate in the helical motif, does not noticeably affect binding. Thus, we show experimentally that an asymmetric charge distribution on an alpha-helix can represent an important element for binding nucleic acids.     

Dissociation and unfolding of bovine odorant binding protein at acidic pH

Dissociation of bovine odorant binding protein (bOBP) dimers to monomers at pH 2.5 has been confirmed through size exclusion chromatography experiments. Moreover, structural and binding properties of the acidic monomer and neutral dimer have been compared using a combination of experimental (circular dichroism and fluorescence) and computational (molecular dynamics) techniques. The secondary and tertiary structures of bOBP are largely maintained at acidic pH, but molecular dynamics simulations suggest the loop regions (N-terminal residues, Omega-loop and C-terminal segments) are more relaxed and Phe36 and Tyr83 residues are involved in the regulation of the binding cavity entrance. The formation of a molten globule state at acidic pH, suggested by the strong enhancement of fluorescence of 8-anilino-1-naphtalenesulphonic acid (ANS), is not confirmed by any significant change in the near UV circular dichroism spectrum. Functionality measurements, deduced from the interaction of bOBP with 1-amino-anthracene (AMA), show that the binding capacity of the protein at acidic pH is preserved, though slightly looser than at neutral pH. Unfolding of acidic bOBP, induced by guanidinium chloride (GdnHCl), was investigated by means of CD spectroscopy, steady state fluorescence, fluorescence anisotropy and light scattering. The stability of the acidic monomer is lower than that of the neutral dimer, owing to the loss of the swapping interactions, but renaturation is completely reversible. Finally, in contrast with the neutral dimer, at low denaturant concentration some aggregation of the acidic monomer, which vanishes before the unfolding transition, has been observed.     

Marked difference between self-aggregations of first and fourth repeat peptides on tau microtubule-binding domain in acidic solution

The heparin-induced self-aggregation behaviours of four repeat peptides (R1-R4) in an acidic solution (pH = 4.5) were investigated by fluorescence and circular dichroism (CD) measurements and compared with those in a neutral solution (pH = 7.5). In contrast with the self-aggregation-resistive behaviours of the R1 and R4 repeat peptides in the neutral solution, the R4 peptide formed a filament similarly to the R2 and R3 peptides in the acidic solution, whereas the R1 peptide still showed resistive behaviour for filament formation. This is the first report on the markedly different self-aggregation behaviours of the first and fourth repeat peptides on tau microtubule-binding domain.     

Deletion of two C-terminal Gln residues of 12-26-residue fragment of melittin improves its antimicrobial activity

In our previous paper it was shown that the two C-terminal Gln residues of a C-terminal 15-residue fragment, Mel(12-26) (GLPALISWIKRKRQQ-NH2), of melittin and a series of individual substituted analogues might not involved in the interaction with bacterial membranes. In this paper, peptides with one and two Gln residues deletion, respectively, Mel(12-25) and Mel(12-24), were synthesized and characterized. Both of the deletion peptides showed higher antimicrobial activities than the parent peptide, Mel(12-26). If both of the Gln residues of Mel(12-26) were respectively replaced by a hydrophilic amino acid Gly, the antimicrobial activity increased slightly. If the Gln residue of Mel(12-25) was replaced by a hydrophobic amino acid Leu, the antimicrobial activity changed little, although the substituted peptide possessed much higher hydrophobicity and higher alpha-helical conformation percentage in 1,1,1,3,3,3-hexafluoro-2-propanol/water determined by circular dichroism spectroscopy (CD) than the parent peptide. These results indicated that the two C-terminal residues might be indeed not involved in the binding to bacterial membranes. The antimicrobial activity increasing with the residue deletion may be caused by the decrease of the translational and rotational entropic cost of the binding of the peptides to bacterial membranes because of the lower molecular weights of the deletion peptides.     

Preparation and in vitro evaluation of novel lipopeptide transfection agents for efficient gene delivery

Gene therapy by delivery of nonviral expression vectors is highly desirable, due to their safety, stability, and suitability for production as bulk pharmaceuticals. However, low transfection efficiency remains a limiting factor in application on nonviral gene delivery. Despite recent advances in the field, there are still major obstacles to overcome. In an attempt to construct more efficient nonviral gene delivery vectors, we have designed a series of novel lipopeptide transfection agents, consisting of an alkyl chain, one cysteine, 1 to 4 histidine and 1 to 3 lysine residues. The lipopeptides were designed to facilitate dimerization (by way of the cysteine residues), DNA binding at neutral pH (making use of charged lysine residues), and endosomal escape (by way of weakly basic histidine residues). DNA/lipopeptide complexes were evaluated for their biophysical properties and transfection efficiencies. The number and identity of amino acids incorporated in the lipopeptide construct affected their DNA/lipopeptide complex forming capacity. As the number of lysine residues in the lipopeptide increased, the DNA complexes formed became more stable, had higher zeta potential (particle surface charge), and produced smaller mean particle sizes (typically 110 nm at a charge ratio of 5.0 and 240 nm at a charge ratio of 1.0). The effect of inclusion of histidines in the lipopeptide moiety had the opposite effect on complex formation to lysine, but was necessary for high transfection efficiency. In vitro transfection studies in COS-7 cells revealed that the efficiency of gene delivery of the luciferase encoding plasmid, pCMV-Luc, mediated by all the lipopeptides, was much higher than poly(L-lysine) (PLL), which has no endosomal escape system, and in two cases was slightly higher than that of branched polyethylenimine (PEI). Lipopeptides with at least two lysine residues and at least one histidine residue produced spontaneous transfection complexes with plasmid DNA, indicating that endosomal escape was achieved by incorporation of histidine residues. These low molecular weight peptides can be readily synthesized and purified and offer new insights into the mechanism of action of transfection complexes.     

Binding of Zn-chlorin to a synthetic four-helix bundle peptide through histidine ligation

We have used a two histidine-containing synthetic peptide (Sharp et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 10465-10470) as a scaffold to bind Zn(II) chlorin e6 (ZnCe6) through histidine ligation. Protocols for the preparation and purification of the peptide using an Escherichia coli expression system are presented. Size-exclusion chromatography and circular dichroism measurements indicate that the peptide self-assembles into a four-helix bundle protein. Two variants of the peptide lacking either one or both of the histidine residues were used to demonstrate the stoichiometry of ZnCe6 binding. Comparison of the titration profiles determined by UV-vis spectroscopy for the purified one- and two-histidine peptides suggests that the two-histidine peptide can bind two ZnCe6. The binding stoichiometry of ZnCe6 was verified by gel chromatography and native gel electrophoresis using the peptide variant lacking histidine residues as the control. Like many other chlorophyll analogue molecules, ZnCe6 can be photooxidized. The light-induced electron transfer between the ZnCe6-peptide complex and the added phenyl-p-benzoquinone was measured using time-resolved EPR spectroscopy and shown to be faster and have a higher yield than the electron transfer between unbound ZnCe6 and quinone. The implications of constructing a ZnCe6-peptide complex in terms of artificial photosynthesis are discussed.     

Probing the interaction between vesicular stomatitis virus and phosphatidylserine

The entry of enveloped animal viruses into their host cells always depends on membrane fusion triggered by conformational changes in viral envelope glycoproteins. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion between the viral envelope and the endosomal membrane at the acidic environment of this compartment. In this work, we evaluated VSV interactions with membranes of different phospholipid compositions, at neutral and acidic pH, using atomic force microscopy (AFM) operating in the force spectroscopy mode, isothermal calorimetry (ITC) and molecular dynamics simulation. We found that the binding forces differed dramatically depending on the membrane phospholipid composition, revealing a high specificity of G protein binding to membranes containing phosphatidylserine (PS). In a previous work, we showed that the sequence corresponding amino acid 164 of VSV G protein was as efficient as the virus in catalyzing membrane fusion at pH 6.0. Here, we used this sequence to explore VSV–PS interaction using ITC. We found that peptide binding to membranes was exothermic, suggesting the participation of electrostatic interactions. Peptide–membrane interaction at pH 7.5 was shown to be specific to PS and dependent on the presence of His residues in the fusion peptide. The application of the simplified continuum Gouy–Chapman theory to our system predicted a pH of 5.0 at membrane surface, suggesting that the His residues should be protonated when located close to the membrane. Molecular dynamics simulations suggested that the peptide interacts with the lipid bilayer through its N-terminal residues, especially Val¹⁴⁵ and His¹⁴⁸. Fabiana A.Carneiro and Pedro A. Lapido-Loureiro contributed equally to this work An erratum to this article can be found at     

Design of a pH-sensitive pore-forming peptide with improved performance.

GALA is a 30 residue synthetic peptide designed to interact with membranes in a pH-sensitive manner, with potential applications for intracellular drug and gene delivery. Upon reduction of the pH from neutral to acidic, GALA switches from random coil to alpha-helix, inserts into lipid bilayers, and forms oligomeric pores of defined size. Its simple sequence and well-characterized behavior make the peptide an excellent starting point to explore the effects of sequence on structure, pH sensitivity, and membrane affinity. We describe synthesis and characterization of two derivatives of GALA, termed GALAdel3E and YALA. GALAdel3E has a deletion of three centrally located glutamate residues from GALA, while YALA replaces one glutamate residue with the unusual amino acid 3,5-diiodotyrosine. Both derived peptides retain pH sensitivity, showing no ability to cause leakage of an encapsulated dye from unilamellar vesicles at pH 7.4 but substantial activity at pH 5. Unlike GALA, neither peptide undergoes a conformational change upon reduction of the pH, remaining alpha-helical throughout. Interestingly, the pH at which the peptides activate is shifted, with GALA becoming active at pH approximately 5.7, GALAdel3E at pH approximately 6.2, and YALA at pH approximately 6.7. Furthermore, the peptides GALAdel3E and YALA show improved activity compared with GALA for cholesterol-containing membranes, with YALA retaining the greatest activity. Improved activity in the presence of cholesterol and onset of activity in the critical range between pH 6 and 7 may make these peptides useful in applications requiring intracellular delivery of macromolecules, such as gene delivery or anti-cancer treatments.     

pH-Dependent conformational changes and topology of a herpesvirus translocating peptide in a membrane-mimetic environment

Pol peptide, an oligopeptide corresponding to the 27 C-terminal amino acids of DNA polymerase from herpes simplex virus type 1, has recently been suggested to translocate from endosomal compartments into the cytosol after being intracellularly delivered via a protein carrier. While an acidic environment was thought to be important for Pol peptide membrane translocation, the mechanism of translocation remains unclear. To investigate the influence of an acidic environment on the conformational properties of the peptide and on its propensity to interact with lipid bilayers, we characterized the structure of Pol peptide at different pH values by both circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. The influence of detergent micelles, which mimic biological lipid membranes, on the peptide secondary structure was also studied. Our CD results indicate that the peptide is in a random conformation in aqueous solution at both acidic and basic pH, whereas in the presence of dodecylphosphocholine (DPC) micelles, it assumes a partial alpha-helical structure which is significantly pH-dependent. An NMR study confirmed that, in the presence of DPC micelles, a short C-terminal alpha-helix is present at pH 6.5, whereas almost two-thirds of the peptide (residues 10-26) fold into an extended amphipathic alpha-helix at pH 4.0. The orientation of Pol peptide relative to the DPC micelle was investigated using paramagnetic probes at both pH 4.0 and 6.5. These studies show that the peptide inserts deeply into the micelle at pH 4.0, whereas it is more exposed to the aqueous environment at pH 6.5. On the basis of these results, a model which might explain the mechanism of translocation of Pol peptide from acidic endosomes to the cytosol is discussed.     

The sterol carrier protein-2 amino terminus: a membrane interaction domain.

Sterol carrier protein-2 (SCP2) is a small, 123 amino acid, protein postulated to play a role in intracellular transport and metabolism of lipids such as cholesterol, phospholipids, and branched chain fatty acids. While it is thought that interaction of SCP2 with membranes is necessary for lipid transfer, evidence for this possibility and identification of a membrane interaction domain within SCP2 has remained elusive. As shown herein with circular dichroism and a direct binding assay, SCP2 bound to small unilamellar vesicle (SUV) membranes to undergo significant alteration in secondary structure. The SCP2 amphipathic N-terminal 32 amino acids, comprised of two alpha-helical segments, were postulated to represent a putative phospholipid interaction site. This hypothesis was tested with a series of SCP2 N-terminal peptides, circular dichroism, and direct binding studies. The SCP2 N-terminal peptide (1-32)SCP2, primarily random coil in aqueous buffer, adopted alpha-helical structure upon interaction with membranes. The induction of alpha-helical structure in the peptide was maximal when the membranes contained a high mole percent of negatively charged phospholipid and of cholesterol. While deletion of the second alpha-helical segment within this peptide had no effect on formation of the first alpha-helix, it significantly weakened the peptide interaction with membranes. Substitution of Leu(20) with Glu(20) in the N-terminal peptide disrupted the alpha-helix structure and greatly weakened the peptide interaction with membranes. Finally, deletion of the first nine nonhelical amino acids had no effect either on formation of alpha-helix or on peptide binding to membranes. N-Terminal peptide (1-32)SCP2 competed with SCP2 for binding to SUV. These data were consistent with the N-terminus of SCP2 providing a membrane interaction domain that preferentially bound to membranes rich in anionic phospholipid and cholesterol.     

Templated assembly of the pH-sensitive membrane-lytic peptide GALA.

Delivery of protein or nucleic acid therapeutics into intracellular compartments may require facilitation to allow these macromolecules to cross otherwise impermeant cellular membranes. Peptides capable of forming membrane-spanning channels hold promise as just such facilitators, although the requirement for peptide oligomerization to form these channels may limit their effectiveness. Synthetic molecules containing multiple copies of membrane-active peptides attached to a template molecule in a pre-oligomerized form have attracted interest for drug-delivery applications. Using three template designs, we synthesized multimeric versions of the pH-sensitive lytic peptide GALA and compared their performance to monomeric GALA. Template assembly stabilized helix formation: templated GALA retained alpha-helical structure even at neutral pH, unlike monomeric GALA. In membrane leakage assays, templated GALA retained the pH sensitivity of the monomer, with improved leakage for dimeric GALA. Surprisingly, trimeric GALA was less effective, particularly when synthesized with a larger and more flexible spacer. Surface plasmon resonance analysis indicated that reversible binding of templated GALA to lipid surfaces at acidic conditions was greatly reduced compared with monomeric GALA, but that the amount of irreversibly bound material was similar. We interpreted these results to indicate that templated peptides may cyclize into 'self-satisfied' oligomeric structures, incapable of further aggregation and subsequent pore formation. Future design of templated peptides must be carefully performed to avoid this unwanted consequence.     

Crotamine mediates gene delivery into cells through the binding to heparan sulfate proteoglycans

Recently we have shown that crotamine, a toxin from the South American rattlesnake Crotalus durissus terrificus venom, belongs to the family of cell-penetrating peptides. Moreover, crotamine was demonstrated to be a marker of centrioles, of cell cycle, and of actively proliferating cells. Herein we show that this toxin at non-toxic concentrations is also capable of binding electrostatically to plasmid DNA forming DNA-peptide complexes whose stabilities overcome the need for chemical conjugation for carrying nucleic acids into cells. Interestingly, crotamine demonstrates cell specificity and targeted delivery of plasmid DNA into actively proliferating cells both in vitro and in vivo, which distinguishes crotamine from other known natural cell-penetrating peptides. The mechanism of crotamine penetration and cargo delivery into cells was also investigated, showing the involvement of heparan sulfate proteoglycans in the uptake phase, which is followed by endocytosis and peptide accumulation within the acidic endosomal vesicles. Finally, the permeabilization of endosomal membranes induced by crotamine results in the leakage of the vesicles contents to the cell cytosol.     

Model peptide‐based system used for the investigation of metal ions binding to histidine‐containing polypeptides

The reaction of histidine‐containing polypeptides with toxic and essential metals and the molecular mechanism of complexation has yet to be determined, particularly with respect to the conformational changes of the interacting macromolecules. Therefore, a system of oligopeptides containing histidine residues in various positions of Ala or Gly sequences has been designed and used in heavy metal comparatively binding experiments. The role of spacing residues (Gly and Ala repeats) in selecting the various conformations was investigated. The newly synthesized peptides and metal ion adducts have been characterized by Fourier transform infrared spectroscopy (FTIR) as well as electrospray ion trap mass spectrometry (ESI–MS) and circular dichroism (CD). The analysis of CD‐spectra of the four peptides in water revealed that the secondary structure depends much on the position of each amino acid in the peptide backbone. Our peptides system reveals various binding mechanisms of metal ions to peptides depending on the position of histidine residue and the corresponding conformations of Ala or Gly sequences. Biological and medical consequences of conformational changes of metal‐bound peptides are further discussed. Thus, the binding of heavy metals to four peptides may serve as a model system with respect to the conformational consequences of the metal addition on the amino acid repeats situated in prion protein. © 2010 Wiley Periodicals, Inc. Biopolymers 93:497–508, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Effective siRNA delivery and target mRNA degradation using an amphipathic peptide to facilitate pH-dependent endosomal escape

Effective delivery of siRNA (small interfering RNA) into the cells requires the translocation of siRNA into the cytosol. One potential delivery strategy uses cell-delivery peptides that facilitate this step. In the present paper, we describe the characterization of an amphipathic peptide that mediates the uptake of non-covalently bound siRNA into cells and its subsequent release into the cytosol. Biophysical characterization of peptide and peptide/siRNA mixtures at neutral and lysosomal (acidic) pH suggested the formation of α-helical structure only in endosomes and lysosomes. Surprisingly, even though the peptide enhanced the uptake of siRNA into cells, no direct interaction between siRNA and peptide was observed at neutral pH by isothermal titration calorimetry. Importantly, we show that peptide-mediated siRNA uptake occurred through endocytosis and, by applying novel endosomal-escape assays and cell-fractionation techniques, we demonstrated a pH-dependent alteration in endosome and lysosome integrity and subsequent release of siRNA and other cargo into the cytosol. These results indicate a peptide-mediated siRNA delivery through a pH-dependent and conformation-specific interaction with cellular membranes and not with the cargo.     

The Arginines in the N-Terminus of the Porcine Circovirus 2 Virus-like Particles Are Responsible for Disrupting the Membranes at Neutral and Acidic pH

Non-enveloped viruses that are endocytosed employ numerous mechanisms to disrupt endosomal membranes for escape into the cellular cytoplasm. These include the use of amphipathic helices or sheets, hydrophobic loops, myristoylated peptides, and proteins with phospholipase activity. Some mechanisms result in immediate deterioration of the endosome, while others form pores in the membrane causing osmolysis to disrupt the endosome and allow viral escape. We describe an additional mechanism by a non-enveloped virus to disrupt endosomal membranes. Porcine circovirus 2 (PCV2) possesses a 41-amino acid arginine-rich motif (ARM) at the N-terminus of its capsid protein that appears to be in the interior of the virus-like particle (VLP). Using in vitro membrane disruption assays, we demonstrate that PCV2 VLP, unassembled capsid, and ARM peptide possess the ability to disrupt endosomal-like membranes, whereas VLP lacking the ARM sequence does not possess this capability. Membrane disruption by VLP is insensitive to pH, but unassembled capsid protein and ARM peptide exhibit diminished activity at low pH. Our liposome disruption assays, circular dichroism, and intrinsic tryptophan fluorescence assays allow us to propose a model for PCV2–endosomal membrane interaction wherein the ARM peptide externalizes from the capsid, its C-terminus (amino acids 28–40) anchors into the membrane, and the arginine-rich N-terminus (amino acids 1–27) drives membrane disruption. To our knowledge, this is the first example of a non-enveloped virus using the arginines of an ARM to disrupt membranes. Also, this is the first example of such study for the Circoviridae family of viruses.