1. Membrane binding and translocation of cell-penetrating peptides
Per E G Thorén, Daniel Persson, Elin K Esbjörner, Mattias Goksör, Per Lincoln, Bengt Nordén Biochemistry. 2004 Mar 30;43(12):3471-89. doi: 10.1021/bi0360049.
Cell-penetrating peptides (CPPs) have been extensively studied during the past decade, because of their ability to promote the cellular uptake of various cargo molecules, e.g., oligonucleotides and proteins. In a recent study of the uptake of several analogues of penetratin, Tat(48-60) and oligoarginine in live (unfixed) cells [Thorén et al. (2003) Biochem. Biophys. Res. Commun. 307, 100-107], it was found that both endocytotic and nonendocytotic uptake pathways are involved in the internalization of these CPPs. In the present study, the membrane interactions of some of these novel peptides, all containing a tryptophan residue to facilitate spectroscopic studies, are investigated. The peptides exhibit a strong affinity for large unilamellar vesicles (LUVs) containing zwitterionic and anionic lipids, with binding constants decreasing in the order penetratin > R(7)W > TatP59W > TatLysP59W. Quenching studies using the aqueous quencher acrylamide and brominated lipids indicate that the tryptophan residues of the peptides are buried to a similar extent into the membrane, with an average insertion depth of approximately 10-11 A from the bilayer center. The membrane topology of the peptides was investigated using an assay based on resonance energy transfer between tryptophan and a fluorescently labeled lysophospholipid, lysoMC, distributed asymmetrically in the membranes of LUVs. By determination of the energy transfer efficiency when peptide was added to vesicles with lysoMC present exclusively in the inner leaflet, it was shown that none of the peptides investigated is able to translocate across the lipid membranes of LUVs. By contrast, confocal laser scanning microscopy studies on carboxyfluorescein-labeled peptides showed that all of the peptides rapidly traverse the membranes of giant unilamellar vesicles (GUVs). The choice of model system is thus crucial for the conclusions about the ability of CPPs to translocate across lipid membranes. Under the conditions used in the present study, peptide-lipid interactions alone cannot explain the different cellular uptake characteristics exhibited by these peptides.
2. Penetratin and related cell-penetrating cationic peptides can translocate across lipid bilayers in the presence of a transbilayer potential
Donato Terrone, Stephane Leung Wai Sang, Liya Roudaia, John R Silvius Biochemistry. 2003 Dec 2;42(47):13787-99. doi: 10.1021/bi035293y.
Fluorescent-labeled derivatives of the Antennapedia-derived cell-penetating peptide penetratin, and of the simpler but similarly charged peptides R(6)GC-NH(2) and K(6)GC-NH(2), are shown to be able to translocate into large unilamellar lipid vesicles in the presence of a transbilayer potential (inside negative). Vesicles with diverse lipid compositions, and combining physiological proportions of neutral and anionic lipids, are able to support substantial potential-dependent uptake of all three cationic peptides. The efficiency of peptide uptake under these conditions is strongly modulated by the vesicle lipid composition, in a manner that suggests that more than one mechanism of peptide uptake may operate in different systems. Remarkably, peptide uptake is accompanied by only minor perturbations of the overall barrier function of the lipid bilayer, as assessed by assays of vesicle leakiness under the same conditions. Fluorescence microscopy of living CV-1 and HeLa cells incubated with the labeled peptides shows that the peptides accumulate in peripheral vesicular structures at early times of incubation, consistent with an initial endosomal localization as recently reported, but gradually accumulate in the cytoplasm and nucleus during more extended incubations (several hours). Our findings indicate that these relatively hydrophilic, polybasic cell-penetrating peptides can translocate through lipid bilayers by a potential- and composition-dependent pathway that causes only minimal perturbation to the overall integrity and barrier function of the bilayer.
3. Application of a novel analysis to measure the binding of the membrane-translocating peptide penetratin to negatively charged liposomes
Daniel Persson, Per E G Thorén, Mattias Herner, Per Lincoln, Bengt Nordén Biochemistry. 2003 Jan 21;42(2):421-9. doi: 10.1021/bi026453t.
The binding of penetratin, a peptide that has been found useful for cellular delivery of large hydrophilic molecules, to negatively charged vesicles was investigated. The surface charge density of the vesicles was varied by mixing zwitterionic dioleoylphosphatidylcholine (DOPC) and negatively charged dioleoylphosphatidylglycerol (DOPG) at various molar ratios. The extent of membrane association was quantified from tryptophan emission spectra recorded during titration of peptide solution with liposomes. A singular value decomposition of the spectral data demonstrated unambiguously that two species, assigned as peptide free in solution and membrane-bound peptide, respectively, account for the spectral data of the titration series. Binding isotherms were then constructed by least-squares projection of the titration spectra on reference spectra of free and membrane-bound peptide. A model based on the Gouy-Chapman theory in combination with a two-state surface partition equilibrium, separating the electrostatic and the hydrophobic contributions to the binding free energy, was found to be in excellent agreement with the experimental data. Using this model, a surface partition constant of approximately 80 M(-)(1) was obtained for the nonelectrostatic contribution to the binding of penetratin irrespective of the fraction of negatively charged lipids in the membrane, indicating that the hydrophobic interactions are independent of the surface charge density. In accordance with this, circular dichroism measurements showed that the secondary structure of membrane-associated penetratin is independent of the DOPC/DOPG ratio. Experiments using vesicles with entrapped carboxyfluorescein showed that penetratin does not form membrane pores. Studies of the cationic peptide penetratin are complicated by extensive adsorption to surfaces of quartz and plastics. By modification of the quartz cell walls with the cationic polymer poly(ethylenimine), the peptide adsorption was reduced to a tolerable level. The data analysis method used for construction of the binding isotherms eliminated errors emanating from the remaining peptide adsorption, which otherwise would prevent a proper quantification of the binding.
