Báo cáo khoa học: Structure and positioning comparison of two variants of penetratin in two different membrane mimicking systems by NMR pdf

Structure and positioning comparison of two variants of penetratin in two different membrane mimicking systems by NMR Mattias Lindberg, Henrik Biversta ˚ hl, Astrid Gra¨ slund and Lena Ma¨ ler Department of Biochemistry and Biophysics, The Arrhenius Laboratories, Stockholm University, Sweden The Antennapedia homeodomain protein of Drosophila has the ability to penetrate biological membranes and the third helix of this protein, residues 43–58, known as penetratin (RQIKIWFQNRRMKWKK-amide) has the same trans- locating properties as the entire protein. The variant, RQI KIFFQNRRMKFKK-amide, here called penetratin (W48F,W56F) does not have the same ability. We have determined a solution structure of penetratin and investi- gated the position of both peptides in negatively charged bicelles. A helical structure is seen for residues Lys46 through Met54. The secondary structure of the variant penetra- tin(W48F,W56F) in bicelles appears to be very similar. Paramagnetic spin-label studies and analysis of NOEs between penetratin and the phospholipids show that pene- tratin is located within the bicelle surface. Penetratin (W48F,W56F) is also located inside the phospholipid bicelle, however, with its N-terminus more deeply inserted than that of wild-type penetratin. The subtle differences in the way the two peptides interact with a membrane in an equilibrium situation could be important for their translocating ability. As a comparison we have also investigated the secondary structure of penetratin(W48F,W56F) in SDS micelles and the results show that the structure is very similar in SDS and bicelles. In contrast, penetratin(W48F,W56F) and penetra- tin appear to be located differently in SDS micelles. This clearly shows the importance of using realistic membrane mimetics for investigating peptide–membrane interactions. Keywords: cell-penetrating peptide; penetratin; pAnt; NMR; bicelle. Cell-penetrating peptides, CPPs, have the ability to trans- locate various cell membranes with high efficiency. If they are covalently linked to a ÔcargoÕ, they still retain their translocating properties, making them suitable for trans- porting large cargoes, such as polypeptides and oligonucleo- tides, across membrane barriers. These properties have made CPPs interesting for use as vectors for delivery of hydrophilic biomolecules and drugs into the cytoplasmic and nuclear compartments of the cell, both in vivo and in vitro [1–3]. The 60 amino acid residue DNA-binding domain of the Drosophila transcription factor translocates membranes. The peptide corresponding to the residues of the third helix of the Antennapedia homeodomain of Drosophila (residues Arg43 through Lys58: RQIKIWFQNRRMKWKK) has been shown to have the same translocating properties as the entire protein [4–7]. The peptide, known as penetratin, has the ability to carry large cargoes, such as oligonucleotides, proteins or other peptides, through biological membranes [1]. Penetratin is a well-studied CPP, both with regards to its translocating properties [5–8] and its induced secondary structure in various membrane mimetic solvents, such as detergent micelles and phospholipid vesicles [9–13]. The translocation process does not seem to require a chiral receptor and the detailed mechanism is still not understood. Knowledge about the interaction between the peptide and the membrane is fundamental for the understanding of the translocation process. Therefore studies in a realistic membrane environment are important. A study of the secondary structure of penetratin in a membrane-like environment with negatively charged SDS micelles has previously been conducted [14] where it was shown that penetratin interacts with the SDS micelle and adopts an a-helical structure in the micelle environment. In a positioning study using paramagnetic probes it was shown that penetratin is located with its most N-terminal residues at the micellar surface and with the C-terminus hidden inside the interior of the micelle [10]. There is evidence that the induced secondary structure of a transport peptide is not an important factor for the transport ability [6,15], but very little is known about the importance of the positioning in the membrane. When changing the two tryptophans (residue 48 and 56) of penetratin to phenylalanines it was shown that the translocating property of penetratin was essentially lost [13]. In this study, we have determined a NMR solution structure of penetratin and investigated the position of penetratin in negatively charged phospholipid bicelles. The secondary structure of the nontranslocating analog, denoted penetratin(W48F,W56F), in negatively charged bicelles has also been investigated together with the position of the peptide relative to the surface and interior of the bicelles. Correpsondence to L. Ma ¨ ler, Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden. Fax: + 46 (0)8155597, E-mail: lena@dbb.su.se Abbreviations: CPP, cell-penetrating peptide; DHPC, 1,2-dihexanoyl- sn-glycero-3-phosphatidylcholine; DMPC, 1,2-dimyristoyl- sn-glycero-3-phosphatidylcholine; DMPG, 1,2-dimyristoyl-sn- glycero-3-phospho-1-glycerol; DMPS, dimyristoyl-sn-glycero- 3-phosphatidylserine; TSPA, 3-trimethylsilyl-propionic acid-d 4 . Note: a web site is available at http://www.dbb.su.se (Received 3 March 2003, revised 11 April 2003, accepted 23 May 2003) Eur. J. Biochem. 270, 3055–3063 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03685.x Bicelles are formed by mixing two phospholipid compo- nents with different chain lengths (e.g. DHPC, dihexanoyl- sn-glycero-phosphocholine and DMPC, dimyristoyl- sn-glycero-phosphocholine) and it has been established that a mixture of DMPC and DHPC produces disk-shaped bicelles [16–18]. The size of the bicelle can be controlled by varying the lipid composition (q ¼ [DMPC]/[DHPC]) and a fraction of the DMPC can be replaced by charged lipids [19]. Smaller isotropic bicelles have been shown to retain their disk-like shape [20,21] properties and are suitable for high-resolution NMR work in which peptides that associate with membranes can be studied [22–26]. Here we present an NMR study of penetratin and the nontranslocating analog in a q ¼ 0.5 bicellar solution with a fraction of the DMPC replaced by the negatively charged phospholipid DMPG. Because penetratin has previously been studied in SDS micelles we also investigated the structure and position of penetratin(W48F,W56F) in SDS. The SDS micelle may be considered as a simple mimic of the amphiphilic environ- ment of a phospholipid bilayer and its dimensions are comparable with those of the peptide, which may influence the interaction between the two and the resulting complex. The phospholipid bicelles are more membrane-like than a SDS micelle and may therefore be a better system for positioning studies of membrane bound peptides. We have compared structure and positioning results obtained using the two types of solvents, micelles and bicelles. Based on our present knowledge we conclude that although secondary structure induction is quite similar in the two solvents, the positioning experiments give a more coherent picture in the bicellar solvent. Experimental procedures Sample preparation Penetratin and penetratin(W48F,W56F) were obtained as HPLC-purified custom syntheses from Neosystem Inc. and were used without further purification. Deuterated SDS was purchased from Cambridge Isotopes Laboratories Inc. Deuterated phospholipids, dihexanoyl-sn-glycero- 3-phosphatidylcholine-d 22 (DHPC), 1,2-dimyristoyl-sn- glycero-3-phosphatidylcholine-d 54 (DMPC), 1,2-dimyris- toyl-sn-glycero-3-phospho-1-glycerol-d 54 (DMPG), 1,2- dimyristoyl-sn-glycero-3-phosphatidylserine-d 54 (DMPS) and the spin-labeled phospholipids 1-palmitoyl-2-stearoyl- sn-glycero-5-doxyl-3-phosphatidylcholine and 1-palmitoyl- 2-stearoyl-sn-glycero-12-doxyl-3-phosphatidylcholine were purchased from Avanti Lipids. The 5- and 12-doxyl stearic acids were obtained from Sigma and the MnCl 2 from Merck. Bicelle samples were produced by mixing a 0.96 M aqueous solution of DHPC with a slurry of the long- chained lipids (DMPC and DMPG or DMPS) in H 2 Oto obtain a sample with a total lipid concentration of 15% (w/v) and q ¼ 0.5, which indicates a bicelle diameter of 80–100 A ˚ [16]. Penetratin was added together with the long- chained lipids to reach a peptide concentration of 3 m M . The pH was checked and adjusted to around 5.5 for each sample and finally, aqueous KCl was added to a final salt concentration of 50 m M . In experiments using paramagnetic probes either small amounts of aqueous solution of MnCl 2 , or small amounts of 5-doxyl- or 12-doxyl-labeled phos- pholipid in methanol-d 4 wasaddedtothesample.Inall NMR samples, 25 lLD 2 O was added for field/frequency locking. The peptide/SDS samples were prepared by dissolving the peptide powder at 2 m M concentration in 300 m M deuterated SDS solution in H 2 O/D 2 O. Under the conditions used, SDS forms stable micelles with an approximate number of 60 SDS molecules per micelle [27]. The H 2 O/ D 2 O-ratio was 90 : 10 and the pH was set to 4.1 by adding small amounts of HCl. The samples used in spin-label experiments were prepared by adding either small amounts of aqueous MnCl 2 , or small amounts of 5-doxyl or 12-doxyl-labeled stearic acid dissolved in methanol-d 4 . Spectroscopy All NMR experiments were performed at 37 or 45 °Con Varian Unity spectrometers operating at 600 MHz 1 H frequency. The chemical shifts were referenced to internal 3-(trimethylsilyl)-propionic acid-2,2,3,3-d 4 (TSPA). Two- dimensional phase-sensitive spectra were collected using the method by States and coworkers [28]. The spectral widths in the two-dimensional experiments were 9000 Hz in both dimensions and typically the number of complex points collected in the x 2 dimension was 4096 and 512 in the x 1 dimension. The data were zero-filled to 8192 points in the x 2 dimension and to 4096 points in the x 1 dimension prior to Fourier transformation. NOESY experiments [29] were recorded with mixing times of 100, 150 and 300 ms and TOCSY experiments [30] were recorded with mixing times of 30, 60 and 90 ms. Water-suppression was achieved with low-power presaturation or with the WATERGATE sequence [31]. The data were processed and analyzed using the VNMR program on a Sun sparc5 work station and with the FELIX software (version 2000, Accelrys Inc.). The chemical shift assignments for penetratin in bicelles and for penetratin(W48F,W56F) in bicelles and SDS have been deposited with the BioMagResBank under accession num- bers 5542 and 5543, respectively. CD measurements were made on a Jasco J-720 CD spectropolarimeter using a 0.01-mm quartz cuvette. Wave- lengths ranging from 190 to 250 nm were measured, with a 0.2-nm step resolution and 100 nmÆmin )1 speed. The temperature was controlled by a PTC-343 controller. Spectra were collected and averaged over four scans. The a-helical content was established from the amplitude at 222 nm, as previously described [32], assuming that only a-helix and random coil conformations contribute to the CD spectrum. Structure calculation of penetratin Distance constraints were generated from quantifying NOESY (s mix ¼ 100 ms) cross-peak intensities according to the procedure previously outlined [25]. The structures were checked against the NOESY data to verify that no short distances had missing NOE cross-peaks in the data. However, this procedure was of limited use due to overlap with strong lipid cross-peaks. A total of 129 distance constraints were identified, mostly sequential and medium- range NOEs defining the secondary structure of the peptide 3056 M. Lindberg et al. (Eur. J. Biochem. 270) Ó FEBS 2003 (40 intraresidue, 54 sequential and 35 medium-range NOEs). For a small peptide like penetratin one does not expect to find many long-range NOEs. The structures were calculated using DYANA [33] version 1.5, using the standard annealing algorithm. A total of 60 structures were calculated and 20 were selected to represent the final solution structure based on their target function and constraint violations. The quality of the structure was checked with the program PROCHECK _ NMR [34] and analyses of secondary structure were performed with the GAP software package [35]. The structures were visualized using INSIGHT (version 2000, Accelrys). The coordinates of the final structures together with the input constraints have been deposited with the PDB under accession number 1QMQ. Results Structure of penetratin in phospholipid bicelles CD measurements were performed in order to establish the effect of different phospholipid compositions on the secon- dary structure of penetratin (Figs 1 and 2). Figure 1 shows CD spectra from penetratin in different bicellar solutions. The CD spectra reveal that the structure of penetratin is mostly random coil in water (Fig. 1a) and that it interacts strongly with the partly charged bicelles to obtain a helical structure (Fig. 1d). There is also some structure induction in neutral bicelles, although to much less extent (Fig. 1c). Furthermore it is evident that the peptide does not interact with DHPC alone to obtain a helical structure (Fig. 1b). Therefore it is safe to assume that penetratin most likely interacts with the charged surface of the negatively charged bicelles while it does not interact with the DHPC rim. There is a slight difference in the structure of penetratin in bicelles containing 10% DMPG and 10% DMPS, and penetratin seems to be more helical in bicelles with DMPG (43% vs. 35%, Fig. 2b). The effect of varying the lipid/peptide ratio (L/P) was also investigated and it was seen that the amount of helical structure increased with higher L/P ratio in agreement with what has previously been observed in phospholipid vesicles [36]. To investigate in more detail the structure of the peptide in the bicelle solvent, a solution structure of penetratin was calculated based on 129 distance constraints. Resonance assignments for all but the two terminal residues were obtained from analysis of TOCSY data and sequential assignments were obtained from NOE connectivities (Fig. 3). Structural statistics for the ensemble of 20 struc- tures are presented in Table 1 and the structure represented by an ensemble of 20 models is shown in Fig. 4. The structure shows low constraint violations and good stereo- chemical properties, with only one residue within the entire ensemble falling in the disallowed region of the Ramachandran plot. Based on analyses of hydrogen bonds and backbone torsion angles, helical structure could be assigned for residues Lys46 through Met54, although Lys46 is not as well-defined and shows weaker hydrogen bonds. Arg53, at the C-terminus of the helix is involved in hydrogen bonds to both Gln50 and Phe49, indicating a mixture of a-helical and 3 10 -helical character. Met54 is only hydrogen bonded to Asn51. Although the helix is somewhat irregular, we are nevertheless able to identify a helical segment in the central part of penetratin. The amount of helix is greater in the structure than that predicted from CD measurements, but this can be explained by an equilibrium between penetratin bound to bicelles and in water, where there is a fast exchange between the two forms. The NMR structure is representative for the bicelle-bound form of penetratin, while CD spectra represent an average over the sample. Structure of penetratin(W48F,W56F) in phospholipid bicelles The nonactive penetratin(W48F,W56F) variant was studied by high resolution NMR in both phospholipid bicelles and SDS micelles. Experiments and assignments were made as described above for penetratin. Secondary chemical shifts for the H a resonances in peptides or proteins carry information on secondary structure [37,38] and they were Fig. 1. CD-spectra for penetratin in different solvents. (a) in buffer; (b) 300 m M DHPC; (c) q ¼ 0.5 DMPC/DHPC bicelles; (d) q ¼ 0.5 DMPC/DMPG/DHPC bicelles ([DMPG]/[DMPC] ¼ 0.1). The tem- perature was 37 °CandthepH5.5. Fig. 2. Effect of surface charge and peptide concentration on the a-helical content in CD-spectra of penetratin for different compositions of the bicelles. In all samples, the ratio of long-chained to short-chained phospholipids was q ¼ 0.5. The temperature was 37 °Candthe pH 5.5. (a) [DMPG]/[DMPC] ¼ 0.1, 1 m M penetratin; (b) [DMPS]/ [DMPC] ¼ 0.1, 1 m M penetratin; (c) [DMPG]/[DMPC] ¼ 0.1, 3 m M penetratin; (d) [DMPG]/[DMPC] ¼ 0.05, 1 m M penetratin. Ó FEBS 2003 Structure and position of penetratin (Eur. J. Biochem. 270) 3057 calculated for penetratin(W48F,W56F) as well as for penetratin according to Sykes and coworkers in both solvents (Fig. 5). The data on penetratin in SDS micelles was taken from [10]. Analysis of the NOESY spectrum for penetratin(W48F,W56F) provided characteristic amide- amide, d aN (i,i +3) and d ab (i,i + 3) NOE cross-peaks indicative of a-helical secondary structure. The NOE results for penetratin(W48F,W56F) and penetratin in the phos- pholipid bicelles are summarized in Fig. 6. Based on the similarity of the chemical shift and NOE data, we conclude that the structure of penetratin(W48F,W56F) should be very similar to that of penetratin in bicelles. The NOE cross-peak patterns for penetra- tin(W48F,W56F) in SDS gave similar evidence of a-helix Fig. 3. NMR data for penetratin in phospholipid bicelles (q = 0.5, [DMPG]/[DMPC] = 0.1). (A)Partofa600-MHzTOCSYspectrum recorded at 37 °CshowingtheH N –H a region; (B) the H N –H N region of a 600-MHz NOESY spectrum (s mix ¼ 100 ms) with the indicated assignments. Table 1. Structural statistics for the ensemble of 20 penetratin structures in bicelles calculated with DYANA . Number of constraints 129 Target function (A ˚ 2 ) 0.04 Maximum distance violation (A ˚ ) 0.07 Backbone rmsd (A ˚ ) All residues 1.24 Residues 45–55 0.64 Ramachandran plot regions (%) Most favored 76.4 Allowed region 18.2 Generously allowed 5.0 Disallowed 0.4 Fig. 4. Solution structure of penetratin in acidic phospholipid bicelles representedbyanensembleof20structures.The overlay was performed by superimposing backbone atoms in residues Ile45-Lys55. Fig. 5. Secondary H a chemical shifts for penetratin and penetra- tin(W48F,W56). (A) penetratin (filled squares) and penetra- tin(W48F,W56) (open squares) in phospholipid bicelles with q ¼ 0.5 and [DMPG]/[DMPC] ¼ 0.1. (B) penetratin (filled circles) and penetratin(W48F,W56F) (open circles) in 300 m M SDS. The data on penetratin in SDS micelles was taken from [10]. 3058 M. Lindberg et al. (Eur. J. Biochem. 270) Ó FEBS 2003 as seen for active penetratin (data not shown). The secondary chemical shifts in SDS indicate that the peptide adopts an a-helical conformation to a larger extent in this solvent. Previous investigations of peptides in bicelles and SDS have shown that SDS can restrain motional flexibility of the peptide, which might lead to a more structured peptide. The position of penetratin with respect to the bicelle Paramagnetic probes were added to the different samples to determine positioning of the peptide relative to the surface and interior of the bicelle and micelle, respectively. In studies of SDS micelles alone, Mn 2+ ions have been shown to affect SDS resonances from nuclei at the surface of the micelle. 5-doxyl- and 12-doxyl-labeled stearic acids were shown to affect resonances from nuclei inside (5-doxyl) or deeply buried (12-doxyl) in the micelle [39]. Interpretation of the paramagnetic probe experiments can be done semiquantitatively by evaluating the loss of amplitude for the cross-peaks of the peptide, by measuring the remaining amplitude. The remaining amplitude [40], RA, is defined as: RA ¼ N Á A paramag: A 0 where A paramag is the amplitude of the crosspeak measured when the paramagnetic agent is added and A 0 is the amplitude with no paramagnetic agent present. N is a normalizing factor in order to normalize the remaining amplitude so that the least affected crosspeak has a remaining amplitude of 100%. Similarly, the position of the peptide relative to the bicelle can be estimated by observing the effect of specific paramagnetic probes on resonances in the NMR spectrum. In the present study we have investigated the effects of Mn 2+ ions as well as of a 5-doxyl- and 12-doxyl-labeled phospholipid on the 1 H resonances of the peptide. The 5-doxyl labeled phospholipid has been shown to insert into phospholipid vesicles with the doxyl group at a distance from the center of the bilayer of 12 A ˚ [41,42]. These measurements are only semiquantitative and we judge that the errors on the remaining amplitudes are on the order of ± 20%. Looking at the lipids, the results show that the paramag- netic probes clearly affect the resonances originating from the phospholipids. The Mn 2+ ions very efficiently remove resonances of protons close to the phosphate head-group, i.e. of the choline CH 2 protons (4.30 p.p.m and 3.68 p.p.m), and the 2-CH glycerol proton (at 5.26 p.p.m), while leaving aliphatic side-chain protons unaffected (data not shown). The 5-doxyl-group clearly affects signals originating from the aliphatic side-chains, as well as glycerol lipid signals, although no visible effect was seen on the methyl resonance. The 12-doxyl-labeled phospholipid has a large broadening effect on the methyl proton resonances as well as on the rest of the aliphatic side-chain, while leaving the choline and glycerol protons, close to the head-group, much less broadened. Curves depicting the effect of the paramagnetic probes on signal intensities for penetratin in bicelles are shown in Fig. 7. The addition of paramagnetic agents in low concentrations does not seem to alter the structure of the peptide or integrity of the bicelles as the penetratin spectrum remained essentially the same. Overall, Mn 2+ ions do not seem to have a specific effect on the penetratin resonances (Fig. 7A) and it is difficult to judge whether there is a general line-broadening effect on the entire peptide or if the peptide is more or less protected from Mn 2+ ions. Resonances from side-chain protons belonging to Gln44, Lys55 and Lys57 disappear in the presence of Mn 2+ already at lower concentrations, 0.25–0.5 m M . NOEs were observed between the H N resonances belonging to residues Gln50, Asn51, Arg53 and Lys55 and the CH 2 choline protons (at 4.30 p.p.m., 3.68 p.p.m). The side-chains of the two tryptophan residues, Trp48 and Trp56 have NOEs to both the 2-CH 2 glycerol protons and to the choline protons (3.68 p.p.m) indicating that the peptide resides within the head-group region of the bilayer, supporting the conclusion that the Mn 2+ ions have a slight general broadening effect on the peptide. The residues most affected by the 5-doxyl-labeled phospholipid are Ile47, Trp48 and Phe49, situated at the N-terminus, and surprisingly Met54. At a concentration of 1m M 5-doxyl spin-label the cross-peaks for Trp48 and Phe49 disappear while the cross-peak for Met54 is greatly reduced (Fig. 7B), and when the spin-label concentration is increased to 2 m M , the cross-peaks for residues Ile45 through Phe49 disappear completely. The results obtained with the 12-doxyl labeled phospho- lipid (Fig. 7C) were similar to those obtained with the lipid labeled at position 5, i.e. the same residues were primarily affected, although to a much less extent, and signal still remained for Ile45 through Ile47 even at a concentration of 2m M . Fig. 6. A summary of amide-amide, d aN (i,i +3), d aN (i,i +4) and d ab (i,i + 3) NOE connectivities. (A) penetratin in q ¼ 0.5 bicelles with [DMPG]/[DMPC] ¼ 0.1 (B) penetratin(W48F,W56) in q ¼ 0.5 bicelles with [DMPG]/[DMPC] ¼ 0.1. Ó FEBS 2003 Structure and position of penetratin (Eur. J. Biochem. 270) 3059 Positioning of penetratin(W48F,W56F) in phospholipid bicelles The same experiments with paramagnetic probes was performed for penetratin(W48F,W56F). The results are summarized in Fig. 7 together with the results obtained for penetratin, and it can be seen that the trends in the results are similar. Notably, there is a significant effect of the Mn 2+ ions on the C-terminal residues Trp56, Lys57 and Lys58, which is not seen in penetratin. In analogy with what was discussed for penetratin, this implies that the N-terminal residues are more protected from Mn 2+ ions than the C-terminus, suggesting that there is a difference from the broadening effect seen for penetratin. The experiments using the 5-doxyl phospholipid (Fig. 7B) show that cross peaks for residues Ile45–Glu50 and Met54 are broadened by the spin label, while most of the C-terminal residues are much less affected. The results from adding 12-doxyl to penetratin(W48F,W56F) (Fig. 7C) show that cross-peaks from residues Ile47, Phe48 and Phe49 completely vanish. Ile45 and Lys46 are less affected but still located near the paramagnetic doxyl group. The C-terminal residues Met54–Lys58 are affected only marginally by this spin label. Positioning of penetratin(W48F,W56F) in SDS micelles Experiments with penetratin(W48F,W56F) in SDS micelles were performed, partly to compare the effects of the two solvent systems and partly to compare with results obtained previously for penetratin in SDS [10]. Figure 8 shows the remaining amplitude of TOCSY H N -H a cross-peaks for penetratin and penetratin(W48F,W56F) in SDS micelles with Mn 2+ ions, 5-doxyl stearic acid and 12-doxyl stearic acid added. These results are not as straightforward to interpret as the results from bicelles. When Mn 2+ ions are added to penetratin(W48F,W56F) in SDS, two residues are more affected than the others, Phe56 and Lys58 (Fig. 8A). At higher Mn 2+ concentrations (1.5 m M ), residues in the C-terminal part are more affected than what is seen in the N-terminal part (data not shown). The results for penetratin(W48F,W56F) with 5-doxyl stearic acid show that the spin label has the largest effect on Phe48 and the on three residues, Lys55, Phe56 and Lys57. Thus it would seem that the same residues are more or less affected by both Mn 2+ and 5-doxyl stearic acid. Finally, adding 12-doxyl stearic acid to penetratin(W48F,W56F) Fig. 7. The remaining amplitude of H N –H a cross-peaks in 600 MHz TOCSY spectra recorded at 45 °Cfor3m M penetratin (closed) and 3m M penetratin(W48F,W56F) (open) in q = 0.5 bicelles with [DMPG]/[DMPC] = 0.1. The paramagnetic agents are (A) 2 m M MnCl 2 (squares) (B) 1 m M 1-palmitoyl-2-stearoyl-sn-glycero-5-doxyl- 3-phosphatidylcholine (circles), and (C) 1 m M 1-palmitoyl-2-stearoyl- sn-glycero-12-doxyl-3-phosphatidylcholine (diamonds). Fig. 8. The remaining amplitude of H N –H a cross-peaks in 600 MHz TOCSY recorded at 45 °Cfor3m M penetratin (closed) and 3 m M penetratin(W48F,W56F) (open) in SDS micelles. The paramagnetic agents are (A) 0.5 m M MnCl 2 (squares) (B) 5 m M 5-doxyl stearic acid (circles), and (B) 5 m M 12-doxyl stearic acid (diamonds). 3060 M. Lindberg et al. (Eur. J. Biochem. 270) Ó FEBS 2003 in SDS micelles shows that two residues, Phe49 and Met54 are most strongly affected. Discussion Penetratin adopts an a-helical structure between residues Lys46 and Met54 in acidic bicellar solution; this structure is similar to the secondary structure of penetratin in SDS micelles [10,14]. Chemical shift and NOE data for pene- tratin(W48F,W56F) indicate that the secondary structure is very similar to the active penetratin in both SDS micelles and phospholipid bicelles. These results suggest that the secondary structure is conserved when the two tryptophan residues are replaced with phenylalanines. The large differ- ence in chemical shift observed for Phe49 in penetratin and in penetratin(W48F,W56F) is most likely due to changes in ring currents when changing amino acid 48 from a tryptophan to a phenylalanine, an effect that is seen in both bicelles and SDS micelles. Hence, we conclude that replacing the two tryptophan residues by phenylalanines, turning penetratin into a nontranslocating peptide does not change the secondary structure. Next, the paramagnetic broadening effects and hence the positioning of the peptide relative to the surface and interior of the phospholipid bicelle were studied for both peptides (Fig. 7). For penetratin, it is difficult to judge from the line- broadening caused by Mn 2+ ions whether the entire peptide is protected from Mn 2+ , or that a more general broadening effect is seen. However, the observed peptide–lipid NOEs, together with the fact that Mn 2+ ions seem to affect the head-group region of the lipids suggest that the peptide resides within the phospholipid head-group layer, at the interface between the head-group region and the hydro- phobic interior. This is supported by the results obtained with the two spin-labeled phospholipids, which show that the hydrophobic residues at the N-terminus are positioned towards the hydrophobic interior of the bicelle. This leads us to believe that penetratin resides more or less parallel to the bicelle surface with its hydrophobic residues interacting with the interior of the bicelle. The results from the spin-label study are mapped on the penetratin structure in Fig. 9, where it is clear that the NOEs and spin-label results are both consistent with the peptide being positioned within the head-group region. Interestingly, subtle differences can be observed for penetratin(W48F,W56F) (Fig. 9). Mn 2+ ions have a more specific effect on the C-terminal residues of this peptide than on penetratin, which implies that part of the peptide is more exposed to Mn 2+ . In addition the 12-doxyl labeled lipid has a greater effect on the N-terminus than the 5-doxyl lipid has, indicating that the N-terminus of pene- tratin(W48F,W56F) inserts somewhat deeper into the bilayer than what is seen for penetratin. This is consistent with the Mn 2+ results and constitutes a significant differ- ence between penetratin and penetratin(W48F,W56F). The N-terminal residues in penetratin(W48F,W56F) seem to be affected by all probes, but to a varying extent indicating a great deal of flexibility, as also suggested by the penetratin structure. In addition one must consider the inherent flexibility of the phospholipids, which adds to the uncertainty in the estimated position. In fact, the 5-doxyl phospholipid and the Mn 2+ both affect protons in the head- group region of the phospholipids, while the 12-doxyl does not. Hence it is not surprising that there is overlap between the areas that these probes measure. Nevertheless, we conclude that both penetratin and penetratin(W48F,W56F) interact with the bicelle surface, as shown by CD, but in slightly different ways. The proposed mechanism for penetratin translocation includes interactions between the charged residues of penetratin and the membrane surface layer, and by substituting the two tryptophans for phenyl- alanines in penetratin, the peptide inserts somewhat deeper into the hydrophobic bilayer, and looses the ability to translocate. Finally, comparison between the position of penetratin and the analog in bicelles and in SDS reveals significant differences. The SDS data are very complex and suggest that the micelle is not a well-defined system in which the peptide is positioned in a simple way. The micelle and its constit- uents are highly flexible and it is clearly seen that several nonterminal residues are affected by more than one paramagnetic probe, especially at the C-terminus of the peptides. These results show that one should be careful when interpreting positioning data from SDS micelles because the size and the curvature of the micelle might force the peptide to a certain position. However, there are also very different charge densities associated with the two membrane mimetic solvents investigated here, which may be important for the positioning experiments with the highly charged peptides. These observations emphasize the import- ance of using realistic membrane substitutes in studies of membrane–peptide interactions, such as positioning inves- tigations. Although the induced secondary structure seems similar in the two solvents, the paramagnetic broadening studies in SDS are much more difficult to interpret and do not entirely agree with what was found in phospholipid bicelles. Conclusions We have determined a solution structure of penetratin in partly charged bicelles. A helical structure is seen for around Fig. 9. The positioning results for penetratin and penetra- tin(W48F,W56F) mapped onto the structure of penetratin. The lines represent the average size of the head-group region of the bilayer. Only effects that result in a remaining amplitude of <0.4 are shown. Blue color indicates the effect of Mn 2+ ions, light red the effect of 1-palmitoyl-2-stearoyl-sn-glycero-5-doxyl-3-phosphatidylcholine, and dark red the effect of 1 m M 1-palmitoyl-2-stearoyl-sn-glycero-12- doxyl-3-phosphatidylcholine. Ó FEBS 2003 Structure and position of penetratin (Eur. J. Biochem. 270) 3061 50% of the peptide (Lys46–Met54). Our results show that penetratin preferentially interacts with the bicellar surface formed by the DMPC/DMPG lipids, as little structure induction is seen in DHPC and in neutral bicelles. Penetr- atin and penetratin(W48F,W56F) are structurally very similar to each other when interacting with phospholipid bicelles. Both peptides are positioned within the bicelle; however, subtle differences in the positioning of the two peptides are seen. Penetratin is not deeply inserted into the lipid bilayer, but seems nevertheless to reside within the bicelle head-group layer, with NOE data suggesting a parallel orientation relative to the surface. The observations further show that the N-terminus of penetratin (W48F,W56F) inserts more deeply into the bicelle as compared to penetratin. This in turn may be the result of a different interaction between the peptide and membrane, affecting its cell-penetrating properties. 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Structure and positioning comparison of two variants of penetratin in two different membrane mimicking systems by NMR Mattias Lindberg, Henrik. property of penetratin was essentially lost [13]. In this study, we have determined a NMR solution structure of penetratin and investigated the position of penetratin