Membrane Interactions of Cell-Penetrating Peptides Probed by Tryptophan Fluorescence and Dichroism Techniques: Correlations of Structure to Cellular Uptake † Christina E. B. Caesar, ‡ Elin K. Esbjo ¨rner, ‡ Per Lincoln, and Bengt Norde ´n* Department of Chemistry and Bioscience, Chalmers UniVersity of Technology, SE-41296 Gothenburg, Sweden ReceiVed October 14, 2005; ReVised Manuscript ReceiVed April 11, 2006 ABSTRACT: This work reports on the binding and conformation of a series of CPPs in the bilayer membranes of large unilamellar vesicles and the effect of the presence of cholesterol. We show a negative correlation between R-helical structure and uptake efficiency for penetratin peptides where the two central arginine residues of penetratin are thought to be important for breaking the secondary structure. Penetratin R-helicity is also reduced upon incorporation of cholesterol into the membrane. Flow linear dichroism in the far-UV region shows that the penetratin peptides adopt a preferential orientation of the R-helix parallel to the bilayer, and the linear dichroism (LD) spectrum in the aromatic region indicates that the tryptophan residues are preferentially oriented parallel to the membrane. The Tat analogue TatP59W and the oligoarginine R 7 W, which are more efficient CPPs than penetratin, bind to membranes as random coils and do not show any orientation in LD, again indicating that R-helicity reduces uptake efficiency. Further, we observe large variations in tryptophan quantum yields for the five CPPs in this study and discuss this in terms of the ability to cause lipid rearrangement. Binding isotherms show that cholesterol increases the affinity of the peptide for the membrane, but tryptophan fluorescence lifetimes are essentially unaltered by incorporation of as much as 40 mol % cholesterol into the membrane, suggesting the absence of specific peptide-cholesterol interactions. Fluorescence emission maxima are insensitive to cholesterol and indicate that the peptide is positioned in the headgroup region. The results on peptide-membrane interactions are discussed in terms of possible uptake mechanisms. Intracellular delivery of large molecules such as oligo- nucleotides and polypeptides for therapeutic applications is hampered due to the obstacle of crossing the hydrophobic plasma membrane. Since the discovery in 1988 that the HIV-1 Tat protein could enter cells (1, 2), cell-penetrating peptides (CPPs) 1 have attracted considerable attention as highly efficient delivery vectors of hydrophilic cargo mol- ecules. Tat peptides together with penetratin, a 16-residue peptide corresponding to the third helix of the Antennapedia homeodomain in Drosophila, are currently among the most commonly investigated CPPs. Tat peptides may vary slightly in sequence, although Vive `s et al. showed that amino acids 48-60 correspond to the most efficient CPP. Deletion of three residues at the C-terminus (PPQ) has an only modest effect on uptake (3), whereas truncation or alanine substitu- tion of any of the charged residues within the basic region of the peptide markedly reduced the rate of uptake (4). The uptake efficiency of Tat was attributed to the guanidinium headgroup of the arginine side chain rather than to positive charge alone since oligomers of arginine exhibited superior internalization characteristics compared to those of corre- sponding lysine, histidine, or ornithine oligomers (4). CPPs were originally thought to cross plasma membranes in a receptor-, energy-, and temperature-independent, non- endocytotic manner (5, 6). Peptide accumulation in the nucleus was often observed (7). The concept of an unknown passive diffusion process for a cationic peptide across the plasma membrane called for thorough investigations, both in vivo and in lipid model systems, for explaining the molecular basis of such a mechanism (8-11). In 2002 and 2003, research on CPP cell uptake took a drastic turn since Lundberg et al. (12), followed by Richard et al. (13), reported that cell fixation, even when using mild conditions, led to artifacts, in both uptake and intracellular distribution of peptides. Subsequent reports have shown that uptake of many CPPs, especially conjugated to cargo molecules, largely follows endocytotic routes. However, CPPs are able to deliver functional cargo, and cell entry, endocytotic or not, † The Swedish Cancer Research Foundation is thanked for financial support. * To whom correspondence should be addressed. E-mail: norden@chembio.chalmers.se. Telephone: +46-31-7723041. Fax: +46- 31-7723858. ‡ These authors contributed equally to this work. 1 Abbreviations: (11,12)-BrPC, 1-palmitoyl-2-stearoyl-11,12-dibromo- sn-glycero-3-phosphocholine; (6,7)-BrPC, 1-palmitoyl-2-stearoyl-6,7- dibromo-sn-glycero-3-phosphocholine; (9,10)-BrPC, 1-palmitoyl-2- stearoyl-9,10-dibromo-sn-glycero-3-phosphocholine; CD, circular dichroism; CPP, cell-penetrating peptide; DA, distribution analysis; DFQP, depth-dependent fluorescence quenching profile; DOPC, 1,2- dioleoyl-sn-glycero-3-phosphatidylcholine; DOPG, 1,2-dioleoyl-sn- glycero-3-phosphatidylglycerol; EDTA, ethylenediaminetetraacetic acid; HEPES, N-(2-hydoxyethyl)piperazine-N′-2-ethanesulfonic acid; LD, linear dichroism; LUV, large unilamellar vesicle; PM, parallax method; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol; TCSPC, time- correlated single-photon counting; TOE, tryptophan octyl ester. 7682 Biochemistry 2006, 45, 7682-7692 10.1021/bi052095t CCC: $33.50 © 2006 American Chemical Society Published on Web 05/25/2006