Interactions of amphipathic CPPs with model membranesMechanisms of translocationConformational investigations and physicochemical approachesPep-1Structural characterization of Pep-1 in solution and in the presence of a SDSStructural characterization of Pep-1 in the presence of phospholipidsStudies at the air-water interface-adsorption at lipid-containing interfacesPenetration into phospholipid vesiclesFormation of carrier/cargo complexes and structural consequencesElectrophysiological measurementsMPGCD investigationsFTIR investigations in the presence of phospholipidsStudies at the air-water interface-adsorption at lipid-containing interfacesConformational investigations in the presence of phospholipidsPolynucleotide binding and induced conformational changesPolynucleotide bindingInduced conformational changesPeptide-induced ionic leakageThe modelsConclusions and perspectivesReferencesARTICLE IN PRESSReviewInteractions of amphipathic CPPs with model membranesSe´bastien Deshayes, May C. Morris, Gilles Divita, Fre´de´ric Heitz*CRBM-CNRS, FRE 2593, 1919, Route de Mende, F-34293 Montpellier cedex, FranceReceived 9 September 2005; received in revised form 7 October 2005; accepted 7 October 2005AbstractWe have investigated the interactions between two carrier peptides and model membrane systems as well as the conformational consequences ofthese interactions. Studies performed with lipid monolayers at the air –water interface have enabled identification of the nature of the lipid– peptideinteractions and characterization of the influence of phospholipids on the ability of these peptides to penetrate into lipidic media. Penetrationexperiments reveal that both peptides interact strongly with phospholipids. Conformational investigations indicate that the lipid –peptide interactiongovern the conformational state of the peptides. Based on the ability of both peptides to promote ion permeabilization of both natural and artificialmembranes, we propose a model illustrating the translocation process. For MPG, it is based on the formation of a h-barrel pore-like structure, whilefor Pep-1, it is based on association of helices.D 2005 Elsevier B.V. All rights reserved.Keywords: Cell-penatrating peptide; Amphipatic peptide; Interactions with membrane; Conformation; Spectroscopic analysisContents1. Mechanisms of translocation ...................................................... 02. Conformational investigations and physicochemical approaches .................................... 02.1. Pep-1 .............................................................. 02.1.1. Structural characterization of Pep-1 in solution and in the presence of a SDS ..................... 02.1.2. Structural characterization of Pep-1 in solution and in the presence of phospholipids ................. 02.1.3. Studies at the air – water interface—adsorption at lipid-containing interfaces ...................... 02.1.4. Penetration into phospholipids vesicles ......................................... 02.1.5. Formation of carrier/cargo complexes and structural consequences ........................... 02.1.6. Electrophysiological measurements ........................................... 02.2. MPG ............................................................... 02.2.1. CD investigations .................................................... 02.2.2. FTIR investigations in the presence of phospholipids ................................. 02.2.3. Studies at the air – water interface – adsorption at lipid-containing interfaces ...................... 02.2.4. Conformational investigations in the presence of phospholipids ............................ 02.3. Polynucleotide binding and induced conformational changes .................................. 02.3.1. Polynucleotide binding ................................................. 02.3.2. Induced conformational changes ............................................ 02.3.3. Peptide-induced ionic leakage ............................................. 03. The models ............................................................... 04. Conclusions and perspectives ...................................................... 0References .................................................................. 00005-2736/$ - see front matter D 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.bbamem.2005.10.004* Corresponding author. Tel.: +33 4 67 61 33 92; fax: +33 4 67 52 15 59.E-mail address: [email protected] (F. Heitz).Biochimica et Biophysica Acta xx (2005) xxx – xxxhttp://www.elsevier.com/locate/bbaBBAMEM-78940; No. of pages: 8; 4C: 6+ modelARTICLE IN PRESSPrimary amphipathic peptides are the result of the sequentialassembly of hydrophobic and hydrophilic domains. The first oneis required for membrane anchoring and for compl ex formationwith hydrophobic cargoes. The hydrophilic domain is requiredto address a subcellular compartment, to improve the solubilityof the vector and for complex formation with hydrophilicnegatively-charged molecules [1].There are three major families of primary amphipathicpeptides: those derived from a signal peptide (SP) [2,3], from afusion peptide (FP) as found in the MPG family [3,4], and thetryptophan-rich sequences forming the Pep family [5]. Thesethree families have a common hydrophilic domain, the NuclearLocalization Sequence (NLS) of SV40 large T antigen:PKKKRKV [6]. All these peptides bear a WSQ sequence whichacts as a linker between the hydrophilic and hydrophobicdomains, thereby maintaining their integrity. The selected SPsequence corresponds to that of the light chain of the immuno-globulin of caiman crocodylus while the FP of MPG was chosenfrom HIV-1 fusion protein gp41 . For Pep peptides, thehydrophobic sequence was selected from the dimerization mot ifat the interface of HIV-1 reverse transcriptase. All of thesepeptides are acetylated and bear a cysteamide group at their N-and C-termini, respectively.GALFLGFLGAAGSTMGAWSQPKKKRKV for MPGKETWWETWWTEWSQPKKKRKV for Pep-1The peptides of the SP family enter cells very rapidly at 4-Cas well as at room temperature. Since endocytosis inhibitorshave no effect on cell entry, the endocytosis pathway has beenruled out to explain the mechanism of internalization. Thesepeptides have been used for the intracellular delivery ofoligonucleotides [3] or porphyrin [7] derivatives which arecovalently linked to the carrier though the cysteamide moiety.However, they have proved to be to xic when u sed atconcentrations