Structure and positioning comparison of two variants of penetratinin two different membrane mimicking systems by NMR Mattias Lindberg, Henrik Biversta˚hl, Astrid Gra¨slund and Lena Ma¨le
Trang 1Structure 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 Penepene-tratin
(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 cargo, 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 NMRsolution 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,
glycero-3-phosphatidylcholine; DMPG,
1,2-3-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)
Trang 2Bicelles 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 (q¼ [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 NMRwork in which peptides that associate
with membranes can be studied [22–26] Here we present an
NMRstudy of penetratin and the nontranslocating analog
in a q¼ 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-d22 (DHPC),
1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine-d54 (DMPC),
1,2-dimyris-toyl-sn-glycero-3-phospho-1-glycerol-d54 (DMPG),
1,2-dimyristoyl-sn-glycero-3-phosphatidylserine-d54 (DMPS)
and the spin-labeled phospholipids
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 MnCl2 from
Merck
Bicelle samples were produced by mixing a 0.96M
aqueous solution of DHPC with a slurry of the
long-chained lipids (DMPC and DMPG or DMPS) in H2O to
obtain a sample with a total lipid concentration of 15%
(w/v) and q¼ 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 mM
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 mM In experiments using paramagnetic
probes either small amounts of aqueous solution of MnCl,
or small amounts of 5-doxyl- or 12-doxyl-labeled phos-pholipid in methanol-d4 was added to the sample In all NMRsamples, 25 lL D2O was added for field/frequency locking
The peptide/SDS samples were prepared by dissolving the peptide powder at 2 mM concentration in 300 mM
deuterated SDS solution in H2O/D2O Under the conditions used, SDS forms stable micelles with an approximate number of 60 SDS molecules per micelle [27] The H2O/
D2O-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 MnCl2, or small amounts of 5-doxyl or 12-doxyl-labeled stearic acid dissolved in methanol-d4 Spectroscopy
All NMRexperiments were performed at 37 or 45°C on Varian Unity spectrometers operating at 600 MHz 1H frequency The chemical shifts were referenced to internal 3-(trimethylsilyl)-propionic acid-2,2,3,3-d4 (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 x2dimension was 4096 and 512 in the x1 dimension The data were zero-filled to 8192 points in the x2 dimension and to 4096 points in the x1dimension 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 theFELIXsoftware (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 (smix¼ 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
Trang 3(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 usingDYANA[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 theGAP 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
310-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 NMRstructure 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 NMRin both phospholipid bicelles and SDS micelles Experiments and assignments were made as described above for penetratin Secondary chemical shifts for the Ha 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 °C and the pH 5.5.
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 °C and the
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.
Trang 4calculated 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, daN(i,i + 3) and dab(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
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) Part of a 600-MHz TOCSY spectrum
recorded at 37 °C showing the H 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
Maximum distance violation (A˚) 0.07
Backbone rmsd (A˚)
Ramachandran plot regions (%)
Fig 4 Solution structure of penetratin in acidic phospholipid bicelles represented by an ensemble of 20 structures The overlay was performed
by superimposing backbone atoms in residues Ile45-Lys55.
Fig 5 Secondary Ha 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].
Trang 5as 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, Mn2+ 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:
RA¼ N Aparamag:
A0
where Aparamagis the amplitude of the crosspeak measured
when the paramagnetic agent is added and A0 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 NMRspectrum In the present study we have investigated the effects of Mn2+ions as well as of a 5-doxyl- and 12-doxyl-labeled phospholipid on the 1H 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 Mn2+ions very efficiently remove resonances of protons close to the phosphate head-group, i.e of the choline CH2protons (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, Mn2+
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 Mn2+ ions Resonances from side-chain protons belonging to Gln44, Lys55 and Lys57 disappear in the presence of Mn2+ already at lower concentrations, 0.25–0.5 mM
NOEs were observed between the HN resonances belonging to residues Gln50, Asn51, Arg53 and Lys55 and the CH2choline 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-CH2glycerol 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 Mn2+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
1 mM 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 mM, 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 phospho-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
2 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.
Trang 6Positioning 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 Mn2+
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 Mn2+ 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 HN-Ha cross-peaks for penetratin and penetratin(W48F,W56F) in SDS micelles with Mn2+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 Mn2+ions are added to penetratin(W48F,W56F) in SDS, two residues are more affected than the others, Phe56 and Lys58 (Fig 8A)
At higher Mn2+concentrations (1.5 mM), 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 Mn2+and 5-doxyl stearic acid Finally, adding 12-doxyl stearic acid to penetratin(W48F,W56F)
Fig 7 The remaining amplitude of HN–Hacross-peaks in 600 MHz
TOCSY spectra recorded at 45 C for 3 m M penetratin (closed) and
3 m 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 C for 3 m 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 12-doxyl stearic acid (diamonds).
Trang 7in 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 Mn2+ions whether the entire peptide
is protected from Mn2+, or that a more general broadening
effect is seen However, the observed peptide–lipid NOEs,
together with the fact that Mn2+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) Mn2+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 Mn2+ 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 Mn2+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 Mn2+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 Mn2+ 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.
Trang 850% 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 penetrPenetr-atin(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 Furthermore, the
results clearly show that phospholipid bicellar solutions
provide suitable membrane substitutes for cell-penetrating
peptides, such as penetratin An important advantage of
using phospholipid bicelles over detergent micelles is
illustrated by the fact that the position of the peptide
relative to the bicelle surface can be investigated in a realistic
membrane-like environment
Acknowledgements
We thank the Swedish NMRCenter, Gothenburg for access to the
NMRspectrometers This study was supported by grants from the
Swedish Science Research Council (to L M and A G.).
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