Báo cáo khoa học: Induction of raft-like domains by a myristoylated NAP-22 peptide and its Tyr mutant potx

Induction of raft-like domains by a myristoylated NAP-22
peptide and its Tyr mutant
Raquel F. Epand
1
, Brian G. Sayer
2
and Richard M. Epand
1,2
1 Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Canada
2 Department of Chemistry, McMaster University, Hamilton, Canada
NAP-22 is a 22-kDa protein found in neurons that is
important for neuronal sprouting and plasticity [1].
In addition to the intact 22-kDa protein, significant
amounts of N-terminal myristoylated fragments of this
protein are also found in many tissues [2]. A protein
with a high sequence homology to NAP-22 and prob-
ably with very similar properties, cortical cytoskeleton-
associated protein (CAP)-23, was first identified by
Widmer and Caroni [3]. Myristoylated proteins are
commonly found in cholesterol-rich domains in mem-
branes [4,5]. Full length NAP-22 partitions into the
low density, detergent-insoluble fraction of neuronal
membranes [6], suggesting its presence in neuronal
rafts. Support for this comes from fluorescence micros-
copy studies using both intact biological membranes
[7,8] as well as model membranes [9]. The protein
binds to liposomes of phosphatidylcholine only when
the bilayer contains high mol fractions of cholesterol
[10,11].
Several proteins with cationic clusters, including
CAP-23 as well as the MARCKS protein and GAP-43,

accumulate in rafts, colocalizing with PtdIns(4,5)P2 [8].
Keywords
cholesterol; domains; differential scanning
calorimetry; MAS ⁄ NMR; phosphatidylinositol
(4,5) diphosphate
Correspondence
R. M. Epand, Department of Biochemistry
and Biomedical Sciences, McMaster
University, Hamilton, ON Canada L8N 3Z5
Fax: +1 905 521 1397
Tel: 1 905 525 9140, extn 22073
E-mail:
(Received 10 December 2004, revised 2
February 2005, accepted 14 February 2005)
doi:10.1111/j.1742-4658.2005.04612.x
The N-terminally myristoylated, 19-amino acid peptide, corresponding to
the amino terminus of the neuronal protein NAP-22 (NAP-22 peptide) is a
naturally occurring peptide that had been shown by fluorescence to cause
the sequestering of a Bodipy-labeled PtdIns(4,5)P2 in a cholesterol-depend-
ent manner. The present work, using differential scanning calorimetry
(DSC), extends the observation that formation of a PtdIns(4,5)P2-rich
domain is cholesterol dependent and shows that it also leads to the forma-
tion of a cholesterol-depleted domain. The PtdIns(4,5)P2 used in the present
work is extracted from natural sources and does not contain any label and
has the native acyl chain composition. Peptide-induced formation of a cho-
lesterol-depleted domain is abolished when the sole aromatic amino acid,
Tyr11 is replaced with a Leu. Despite this, the modified peptide can still
sequester PtdIns(4,5)P2 into domains, probably because of the presence of a
cluster of cationic residues in the peptide. Cholesterol and PtdIns(4,5)P2 also
modulate the insertion of the peptide into the bilayer as revealed by

1
H
NOESY MAS ⁄ NMR. The intensity of cross peaks between the aromatic
protons of the Tyr residue and the protons of the lipid indicate that in the
presence of cholesterol there is a change in the nature of the interaction of
the peptide with the membrane. These results have important implications
for the mechanism by which NAP-22 affects actin reorganization in neurons.
Abbreviations
DH
cal
, calorimetric enthalpy; Bodipy-TMR-PI(4,5)P2, BODIPY TMR-X C
6
-phosphatidylinositol 4,5-diphosphate; CAP-23, cortical cytoskeleton-
associated protein (a protein expressed in chicken having a high degree of homology to NAP-22); DP, direct polarization; DSC, differential
scanning calorimetry; LUV, large unilamellar vesicle; MAS, magic angle spinning; NAP-22 peptide, the myristoylated amino terminal 19
amino acids of NAP-22 (myristoyl-GGKLSKKKKGYNVNDEKAK-amide); NAP-22, neuronal axonal membrane protein, also referred to as brain
acid soluble protein 1 (BASP1 protein), a 22 kDa myristoylated protein; PC, phosphatidylcholine; PO, 1-palmitoyl-2-oleoyl; PtdIns(4,5)P2,
L-a-phosphatidylinositol-4,5-bisphosphate from porcine brain; SO, 1-stearoyl-2-oleoyl; T
m
, transition temperature.
1792 FEBS Journal 272 (2005) 1792–1803 ª 2005 FEBS
The importance of electrostatic interactions in the
sequestering of PtdIns(4,5)P2 by proteins with a cationic
domain has been demonstrated [12]. We have also dem-
onstrated the loss of ability of the NAP-22 peptide to
sequester Bodipy-labeled PtdIns(4,5)P2 in the presence
of high salt concentration [13]. In that work we also
demonstrate specificity of the NAP-22-peptide for
Bodipy-labeled PtdIns(4,5)P2 compared with Bodipy-
labeled PtdIns(3,5)P2 [13]. In addition, using total inter-

nal reflectance fluorescence microscopy, we have shown
that the sequestering of Bodipy-labeled PtdIns(4,5)P2
into domains can be a cholesterol-dependent pheno-
menon [13]. This was demonstrated using a myristoylated
N-terminal peptide of NAP-22, Myristoyl-GGKLSK
KKKGYNVNDEKAK-amide. It is known that in vivo,
in addition to the intact NAP-22 protein, a significant
amount of myristoylated N-terminal fragments of this
protein are also present [2], indicating that the myristo-
ylated N-terminal peptide of NAP-22, such as that used
in this work, is also found physiologically. In the present
work we demonstrate that not only does cholesterol
affect the ability of the NAP-22-peptide to induce the
formation of PtdIns(4,5)P2 domains, but it also causes
the rearrangement of cholesterol leading to the forma-
tion of cholesterol-depleted domains. We also test the
role of the aromatic amino acid residue of the peptide in
these phenomena. In addition we show that cholesterol
also affects the arrangement of the peptide in the bilayer.
The present study uses PtdIns(4,5)P2 from porcine
brain, a natural form that has long acyl chains
enriched in arachidonic acid, and it also does not con-
tain any fluorescent probes. Although PtdIns(4,5)P2
from natural sources is highly enriched in arachidonoyl
groups that should not interact well with liquid
ordered domains of rafts, this lipid nevertheless is
found in raft domains of biological membranes [14].
Results
Differential scanning calorimetry (DSC)
We determined the phase transitions of SOPC and

mixtures of this lipid with one or more of the fol-
lowing components: cholesterol, PtdIns(4,5)P2 and
NAP-22-peptide, using differential scanning calori-
metry (DSC). For each sample, six consecutive DSC
scans were run, three heating scans and three cooling
scans at a scan rate of 2 °CÆmin
)1
. Sequential heating
and cooling scans were reproducible. In the absence of
cholesterol a prominent transition is observed in the
region 0–10 °C, corresponding to the chain melting
transition of SOPC. This transition is better resolved
in cooling than in heating scans, since in some cases
the heating scans, initiated at 0 °C, had not reached a
steady-state baseline in the temperature range of the
transition. The transition of POPC would have been
even more difficult to measure, although POPC was
used for the NMR experiments (see below) because the
NMR results could be more directly compared with
our earlier observations on other systems and to avoid
any artefacts that may result from storing peptide-lipid
mixtures that could attain the gel phase. Nevertheless,
we would expect that these two lipids, SOPC and
POPC, that differ only by two CH
2
groups on one of
the acyl chains, would interact almost identically with
peptides. One of the three cooling scans is presented
for samples of different compositions (Fig. 1A). In the
presence of 40 mol% cholesterol, the chain melting

transition of SOPC is broadened and the enthalpy
lowered (Fig. 1B). We also studied the role of the sole
aromatic amino acid, Tyr, of the NAP-22-peptide by
replacing it with Leu. The temperatures and enthalpies
for the phospholipid chain melting transition are
shown (Table 1). The temperature of the transition is
shifted slightly among the different samples and is low-
ered by the presence of peptide. This is probably a
result of the peptide partitioning more favorably into
the liquid-crystalline phase than into the gel phase. In
addition, the enthalpy of this transition in the presence
of cholesterol, PtdIns(4,5)P2 and the NAP-22-peptide
is increased almost threefold. This indicates that cho-
lesterol has been depleted from a domain of the mem-
brane that can now undergo a more cooperative and
endothermic transition, more like that of the pure
phospholipid. Estimates of the transition enthalpy of
mixtures containing cholesterol have a higher error
because of the low temperature and broadness of the
transition. In addition to the phospholipid transition,
some samples also exhibit a transition corresponding
to the polymorphic transition of anhydrous cholesterol
crystals, which appears in the cooling scans at 21 °C.
The enthalpy and temperature of this transition was
estimated from both cooling and heating scans where
this transition occurs at 38 °C. The temperature differ-
ence between the heating and cooling curves is charac-
teristic of this transition and is caused by the slow rate
of interconversion of two forms of anhydrous choles-
terol crystals [15]. The polymorphic transition of anhy-

drous cholesterol crystals is most clearly seen by DSC
in heating scans. We present examples of heating scans
of either SOPC ⁄ cholesterol (60 : 40) or SOPC ⁄ choles-
terol (50 : 50) containing either 10 or 20 mol% of
the NAP-22 peptide or the Y11L NAP-22 (Fig. 2). The
transition enthalpies of these peaks, obtained from the
areas of the peaks, provide an estimate of the amount
of crystalline cholesterol (Table 2). Pure anhydrous
R. F. Epand et al. Cholesterol-dependent lipid rearrangement
FEBS Journal 272 (2005) 1792–1803 ª 2005 FEBS 1793
Fig. 1. DSC cooling scans. (A) SOPC alone (curve 1) and SOPC
with 0.2 mol% PtdIns(4,5)P2 added (curve 2); 0.2 mol%
PtdIns(4,5)P2 and 10 mol% NAP-22-peptide added (curve 3);
10 mol% NAP-22-peptide added (curve 4). (B) SOPC ⁄ cholesterol
60 : 40 with 0.2 mol% PtdIns(4,5)P2 and 10 mol% NAP-22-peptide
added (curve 1); SOPC ⁄ cholesterol 60 : 40 with 10 mol% NAP-22-
peptide added (curve 2); SOPC ⁄ cholesterol 60 : 40 with 0.2 mol%
PtdIns(4,5)P2 added (curve 3); SOPC ⁄ cholesterol 60 : 40 (curve 4);
SOPC ⁄ cholesterol 60 : 40 with 10 mol% mutant Y11L-NAP-22-pep-
tide added (curve 5) and SOPC ⁄ cholesterol 60 : 40 with 0.2 mol%
PtdIns(4,5)P2 and 10 mol% mutant Y11L-NAP-22-peptide added
(curve 6); Scan rate 2°Æmin
)1
.
Table 1. DSC Transition of SOPC. Transitions observed in cooling
scans at 2°Æmin
)1
of SOPC with additional components listed in the
first three columns. When cholesterol is present it is at a 6 : 4 molar
ratio of SOPC:cholesterol PtdIns(4,5)P2 is at 0.2% of total lipids,

while NAP-22-peptide is 10 mol% of total lipids when present.
Additional components
DH
(kcalÆmol
)1
)
Cholesterol PtdIns(4,5)P2 Peptide T
m
(°C)
- – None 6 4.0
- + None 4.8 4.6
- + NAP-22 peptide 1.7 4
- – NAP-22 peptide 1.7 3.2
+ – None 6 0.35
+ + None Broad
Transition
+ – NAP-22 peptide 0.7 0.36
+ + NAP-22 peptide 1.6 0.85
+ – Y11L mutant No transition
observed
+ + Y11L mutant 0.8 0.37
Fig. 2. DSC heating scans. (A) NAP-22 peptide. (B) Y11L-NAP-22-
peptide. Curve 1, SOPC ⁄ cholesterol 60 : 40 with 10 mol% peptide;
Curve 2, SOPC:cholesterol 60 : 40 with 20 mol% peptide; Curve 3,
SOPC ⁄ cholesterol 50 : 50 with 10 mol% peptide; Curve 4,
SOPC ⁄ cholesterol 50 : 50 with 20 mol% peptide. Scan rate
2°Æmin
)1
.
Cholesterol-dependent lipid rearrangement R. F. Epand et al.

1794 FEBS Journal 272 (2005) 1792–1803 ª 2005 FEBS
cholesterol crystals have an enthalpy of 910 calÆ mol
)1
[16]. In some cases the height of the transition peak is
not proportional to the area because the peaks differ
in their breadth (cooperativity). At SOPC ⁄ cholesterol
(60 : 40) it is clear that the NAP-22 peptide promotes
the formation of a larger amount of cholesterol crys-
tals than does the Y11L mutant peptide. However, at
SOPC ⁄ cholesterol (50 : 50) the difference between the
two peptides in this regard largely disappears. In the
absence of peptide (pure SOPC ⁄ cholesterol 60 : 40 or
50 : 50), no peak is observed corresponding to the
formation of cholesterol crystallites (not shown), but
SOPC ⁄ cholesterol (50 : 50) is close to the solubility
limit of cholesterol [17].
Fluorescence quenching
Addition of the Y11L-NAP-22-peptide to large uni-
lamellar vesicles (LUVs) containing 0.1 mol% Bodipy-
TMR-PI(4,5)P2 results in quenching of the Bodipy
fluorescence (Fig. 3). Self-quenching of the fluorescence
of Bodipy-TMR-PI(4,5)P2 by the MARCKS peptide has
been shown to be caused by sequestering of the labeled
lipid into domains [12]. We show that the quenching
of the Bodipy fluorescence by the Y11L-NAP-22-pep-
tide is not significantly affected by cholesterol (Fig. 3),
unlike the case of the unmodified NAP-22-peptide
[13] that is shown in this figure for comparison. In
addition, in the presence of cholesterol the native
sequence with Tyr is more potent than the Y11L-

NAP-22-peptide in causing quenching of the Bodipy-
TMR-PI(4,5)P2.
NMR
The
1
H NMR spectra of various lipid mixtures in the
presence of 10 mol% of the NAP-22-peptide show
predominantly the resonances of the protons of
POPC (Fig. 4). Because they are well resolved from
other peaks, very small peaks arising from the aroma-
tic protons of Tyr can also be seen in the region of
7 p.p.m. The chemical shifts of these, as well as the
major resonances from the phospholipid are summar-
ized in Table 3. Each of the aromatic peaks was split
into a doublet with a vicinal coupling constant of
7.5 Hz.
Table 2. DSC transition of anhydrous cholesterol crystallites. Tran-
sitions observed in heating scans at 2°Æmin)1 of SOPC with 40 or
50 mol% cholesterol, as well as with added peptide.
%
Cholesterol Peptide
DH
(calÆmol cholesterol
)1
)
40 10% NAP-22 peptide 20
40 20% NAP-22 peptide 33
40 10% Y11L mutant peptide 0
40 20% Y11L mutant peptide 12
50 10% NAP-22 peptide 74

50 20% NAP-22 peptide 130
50 10% Y11L mutant peptide 97
50 20% Y11L mutant peptide 110
Fig. 3. Quenching of the fluorescence emission by the NAP-22-pep-
tide (dashed lines) or by the Y11L-NAP-22-peptide (solid lines) of
Bodipy-TMR-PI(4,5)P2. LUVs composed of POPC with added NAP-
peptide (h) or Y11L-NAP-22-peptide (d). POPC with 40 mol% cho-
lesterol with added NAP-peptide (n) or Y11L-NAP-22-peptide (.).
Maximum emission intensity at 571 nm is plotted against the pep-
tide to lipid molar ratio (P ⁄ L). LUVs were present in the cuvette at
a concentration of 50 l
M and the Bodipy-labelled lipids were pre-
sent as 0.1 mol percentage of the total lipid.
Fig. 4. 1-D
1
H MAS ⁄ NMR spectra of several lipid mixture (as indi-
cated on the right of each spectrum) and also containing 10 mol%
NAP-22-peptide. PC, POPC. See Table 3 for assignments.
R. F. Epand et al. Cholesterol-dependent lipid rearrangement
FEBS Journal 272 (2005) 1792–1803 ª 2005 FEBS 1795
Static
31
P NMR powder patterns demonstrated that
all of the samples used for magic angle spinning
(MAS) studies were in bilayer arrangement (not
shown). Two-dimensional
1
H MAS NOESY spectra
were recorded at 25 °C for four lipid samples, each
with 10 mol% NAP-22-peptide. The lipid component

was either POPC; POPC with 0.2 mol% PtdIns(4,5)P2;
POPC ⁄ cholesterol (6 : 4); POPC ⁄ cholesterol ⁄ PtdIns(4,
5)P2 (60 : 40 : 0.2). No resonances assignable to cho-
lesterol could be detected either with or without the
peptide, in agreement with earlier observations [18].
The peptide is in relatively low concentration and many
of its resonances would not be well resolved from
those of the lipid, except for the Tyr aromatic protons.
We have focused on the relative strength of the NOE
interactions between the Tyr aromatic protons and
other atoms. Stronger NOE interactions between two
atoms are a measure of their closer approach. These
are observed as peaks in the 2D NOESY spectra. Sli-
ces of the NOESY at the resonance position of the
aromatic protons are shown for several lipid mixtures
containing 10 mol% NAP-22-peptide using a delay
time of 50 or 300 ms (Fig. 5). The longer delay times
can result in larger NOEs by allowing more complete
energy transfer through dipolar interactions. However,
longer delay times can also allow NOE effects to be
observed between two groups that are not physically
close to each other as a result of spin diffusion. It is
likely, however, that at least with a 50-ms delay time,
spin diffusion does not contribute greatly to the
observed dipolar interactions [19]. Qualitatively one
can conclude that the aromatic residue of the peptide
inserts into the bilayer with all of the lipid mixtures, as
indicated by the fact that most of the protons of the
phospholipid show cross-peaks with the aromatic pro-
tons. In addition, the presence of cholesterol allows a

closer proximity of the Tyr side chain of the peptide
with the terminal methyl group of the acyl chain of
the lipid as shown by the observation that the intensity
of the cross-peak with the terminal CH
3
group (at
1 p.p.m.) relative to that of the CH
2
resonances at
1.4 p.p.m. is larger in the presence than in the absence
of cholesterol (Fig. 5).
In order to specifically assess how PtdIns(4,5)P2
affects the location of the Tyr residue of the NAP-
22-peptide in the membrane, we calculated difference
spectra by taking a pair of spectra that were identical
except for the presence of PtdIns(4,5)P2. Prior to sub-
traction the two spectra were adjusted for small dif-
ferences in intensity and resonance position so as to
visually give the maximal overlap of the two spectra.
Difference spectra were calculated for pairs of spectra
with PtdIns(4,5)P2 minus the spectra for the same
lipid mixture without PtdIns(4,5)P2 using a delay
time of 50 ms (Fig. 6) or 300 ms (Fig. 7). Slices from
the 2D NOESY spectrum at the two resonance posi-
tions for the aromatic residues for pairs of samples
with or without cholesterol are shown. Peaks of
higher intensity, such as the aromatic peaks at 6.9
and 7.2 p.p.m., the HDO peak at 4.8 p.p.m. and the
quaternary ammonium peak at 3.3 p.p.m. show some
residual intensity in the difference spectra, that we do

not consider significant because the intensity of the
Table 3. Assignment of
1
H NMR resonances.
Assignment
a
Chemical Shift (p.p.m.)
Meta phenolic CH 7.2
Ortho phenolic CH 6.9
Glycerol C2 5.4
HDO 4.8
Glycerol C3 4.5
Choline a 4.4
Glycerol C1 4.1
Choline b 3.7
Quaternary CH
3
3.3
CH
2
CO 2.4
CH
2
CCO 1.7
CH
2
1.4
CH
3
1.0

a
Groups correspond to POPC, except for HDO that are the residual
protons of the water and the phenolic CH of the Tyr aromatic pro-
tons from the NAP-22-peptide.
Fig. 5. 1D slices from the MAS
1
H NOESY
spectrum at the chemical shifts of the aro-
matic protons using a mixing time of 50 and
300 ms. The slice at 7.2 p.p.m. corresponds
to the meta CH of Tyr and that at 6.9 p.p.m.
to the phenolic ortho CH.
Cholesterol-dependent lipid rearrangement R. F. Epand et al.
1796 FEBS Journal 272 (2005) 1792–1803 ª 2005 FEBS
difference spectra peaks represent a small fraction of
the original peak and may arise from imperfect align-
ment of the two spectra. In most cases, these reson-
ance positions show closely spaced peaks of positive
and negative sign, indicating a small difference in
chemical shift between the two spectra. However,
with cholesterol, the difference spectra using a 50-ms
delay time clearly shows several positive peaks in the
region 1–2 p.p.m. (Fig. 6, left). This indicates that in
the presence of cholesterol, PtdIns(4,5)P2 allows a
closer proximity of the Tyr side chain of the peptide
with the methylene groups of the acyl chains of the
lipid. This phenomenon is not observed in the
absence of cholesterol (Fig. 6, right). However, for
the samples without cholesterol, for the slice at
6.9 p.p.m., the difference spectra shows a decrease of

intensity at the resonance position of the CH
2
groups,
compared to the samples with cholesterol, and an
increase of the peak intensity at the resonance posi-
tion of the terminal methyl group at 1 p.p.m. This is
particularly clear from the spectra using a 300-ms
delay time (Fig. 7, lower right spectrum). This indi-
cates the ortho protons of the Tyr side chain gain
closer approach to the terminal methyl groups of the
acyl chains, on a millisecond time scale, in the pres-
ence of PtdIns(4,5)P2, but not cholesterol. It should
be pointed out, however, that there could be contri-
butions to the weaker signals in the difference spectra
from cross-peaks between the aromatic protons and
aliphatic protons of the peptide that are not well
resolved in the 1D spectrum. Even if there was such
a contribution, the results would still indicate that
PtdIns(4,5)P2 affects the geometrical relationship
between the peptide and lipid.
Peptide-induced changes in the chemical shift of the
carbon atoms as measured by
13
C direct polarization
(DP) ⁄ MAS indicate that the peptide affects the chem-
ical shift at many positions in the lipid molecule.
Such shifts are usually interpreted in terms of ring-
current effects caused by the aromatic group of the
peptide. However, it is unlikely that similar ring-
current effects could occur at both the glycerol C3

and terminal methyl group of the acyl chain in the
absence of cholesterol or at the glycerol C2 and the
cholesterol C18 in the presence of cholesterol
(Table 4). We suggest that in addition to ring-current
effects there are peptide-induced changes in lipid
packing and interaction with water. It is known that
dehydration will cause an upfield chemical shift of
13
C resonances [20].
Fig. 6. Calculated differences of spectra shown in Fig. 4 using
50 ms delay time. Difference of spectra with PtdIns(4,5)P2 minus
the spectra of the same mixture without PtdIns(4,5)P2. Pairs of
spectra are either from samples with cholesterol (+ cholesterol) or
without cholesterol (– cholesterol). Resonance position of the slice
is indicated on the graph.
Fig. 7. Same as Fig. 6 but for data with 300 ms delay time.
R. F. Epand et al. Cholesterol-dependent lipid rearrangement
FEBS Journal 272 (2005) 1792–1803 ª 2005 FEBS 1797
Discussion
In addition to electrostatic interactions with PtdIns(4,
5)P2, the NAP-22-peptide has two features that can
contribute to its interaction with membranes. These
features include membrane interactions of the N-ter-
minal myristoyl group and the phenolic side chain of
the Tyr residue, both of which are hydrophobic moiet-
ies known to partition into membranes [21,22].
With regard to myristoylation of NAP-22, this post-
translational modification has been found to be
required for the interaction of this protein with mem-
branes [23]. In addition, the protein has no hydropho-

bic segment and its free energy of partitioning into
membranes can be accounted for by the insertion of its
myristoyl group [11]. Myristoylated proteins are often
found to sequester to cholesterol-rich domains in bio-
logical membranes. We suggest that this group contri-
butes to the cholesterol modulation of the membrane
interaction of the NAP-22-peptide.
We have directly tested the role of the Tyr residue in
the membrane interactions of the NAP-22-peptide by
comparing it with a myristoylated peptide in which the
sole Tyr residue was substituted with Leu. The NAP-
22-peptide is more effective in sequestering cholesterol
than is the Y11L mutant. From the DSC results, this
is indicated by fact that the NAP-22-peptide is able
to promote the formation of a greater cholesterol-
depleted domain as shown by the higher enthalpy of
the SOPC transition in the presence of this peptide
compared with the Y11L-NAP-22-peptide, both in the
presence and absence of PtdIns(4,5)P2 (Table 1). In
addition, in mixtures of SOPC ⁄ cholesterol (60 : 40) the
NAP-22-peptide induces the formation of more anhy-
drous cholesterol crystals than the Y11L mutant
(Table 2). We suggest that these crystals form because
cholesterol surpasses its solubility limit in the mem-
brane in cholesterol-rich domains whose formation is
promoted by the peptides. It should also be pointed
out that any cholesterol that is directly bound to a
peptide would be less likely to form crystals. However,
the amount of cholesterol is much larger than the
amount of peptide, so that most of the cholesterol in

these domains will not be binding directly to the pep-
tide. The Y11L-NAP-22-peptide is slightly less effective
than the NAP-22-peptide in sequestering Bodipy-
TMR-PI(4,5)P2 in the presence of cholesterol (Fig. 3),
but more dramatic is that the cholesterol dependence
of Bodipy-TMR-PI(4,5)P2 sequestering is almost com-
pletely eliminated. Tyr is an essential element in the
CRAC motif, suggested to be responsible for choles-
terol recognition [24]. Although the NAP-peptide does
not have other elements required for a CRAC motif,
the sole presence of an aromatic residue may be a con-
tributing factor for cholesterol interaction. We have
previously shown that the aromatic side chains of the
Table 4. Peptide-induced
13
C chemical shift differences of lipid resonances. Data show the chemical shift differences in p.p.m. for the indi-
cated lipid mixture between the pure lipid and lipid with 10 mol% NAP-22 peptide. Cholesterol present in equimolar ratio with POPC and
PtdIns(4,5)P2 as 0.2 mol%. ND, Not determined because of poor resolution of the peak.
Assignment
Chemical shift
(p.p.m.) POPC
POPC +
PtdIns(4,5)P2
POPC ⁄ cholesterol
(1 : 1)
POPC ⁄ cholesterol
(1 : 1) + PtdIns(4,5)P2
Acyl C ¼ O 174 0.07 0.03 ) 0.10 ) 0.07
Acyl C ¼ C 130.0 0.07 0.04 ) 0.07 ) 0.05
Acyl C ¼ C 129.6 0.08 0.05 0.05 0.04

Glycerol C2 71 0.04 0.00 ) 0.15 ) 0.11
Choline b 67 0.04 0.04 ) 0.02 ) 0.02
Glycerol C3 64 0.08 0.10 0.05 ND
Glycerol C1 63 0.05 0.00 0.00 ND
Choline a 60 0.06 0.03 ) 0.01 ) 0.04
Cholesterol C14 ⁄ 17 57 – – ) 0.03 0.00
Quaternary CH
3
54.5 0.03 0.01 ) 0.04 ) 0.03
Cholesterol C9 51 – – ND 0.03
Cholesterol C13 ⁄ C4 43 – – ) 0.03 ) 0.01
Cholesterol C10 37 – – 0.02 0.00
Acyl C2 35 0.06 0.05 ) 0.06 0.01
Cholesterol C25 28.5 – – ) 0.06 ) 0.04
Cholesterol C19 20 – – ) 0.04 ) 0.04
Cholesterol C21 19.5 – – ) 0.05 ) 0.01
Acyl terminal methyl 14 0.08 0.07 0.03 ) 0.02
Cholesterol C18 13 – – ) 0.14 ) 0.15
Cholesterol-dependent lipid rearrangement R. F. Epand et al.
1798 FEBS Journal 272 (2005) 1792–1803 ª 2005 FEBS
peptide N-acetyl-LWYIK-amide can interact with the
A ring of cholesterol [25]. The Y11L-NAP-22-peptide
is also less potent in inducing the formation of choles-
terol clusters than is the NAP-22-peptide. This is indi-
cated by the observation that no cholesterol crystallites
are observed with SOPC and 40 mol% cholesterol in
the presence of Y11L-NAP-22-peptide, while they do
form in the presence of the NAP-22 peptide. In addi-
tion, there is no evidence for the formation of a choles-
terol-depleted phase with the Y11L-NAP-22-peptide,

which would result in a more cooperative chain melt-
ing transition of SOPC with higher enthalpy (Table 1
and Fig. 1).
Peptides with cationic clusters, even simple oligo-
mers of Lysine, will sequester the polyanionic
PtdIns(4,5)P2 [12,26–28]. The unique feature of the
NAP-22 peptide is that this clustering of PtdIns(4,5)P2
is strongly dependent on the presence of cholesterol
[13]. A well studied peptide that does not require cho-
lesterol for sequestering PtdIns(4,5)P2 is the MARCKS
peptide [12]. There are several differences between
the MARCKS peptide and the NAP-22-peptide. The
MARCKS peptide has 13 positive charges compared
to only seven cationic residues for the NAP-22-peptide.
As a consequence, electrostatic interactions alone will
provide a stronger driving force for the MARCKS
peptide to sequester PtdIns(4,5)P2, compared with the
NAP-22-peptide. Although the MARCKS protein, like
NAP-22, is N-terminally myristoylated, the longest
cluster of five Lys residues in MARCKS begins at resi-
due 86, far removed from the amino-terminal myris-
toyl group. Also the model MARCKS peptide is not
myristoylated, unlike the peptides used in the present
work. With regard to aromatic residues, the MARCKS
peptide has five Phe residues while the NAP-22-peptide
has only one Tyr. In the case of MARCKS peptide,
the major cross-peak between the aromatic resonance
of the peptide and the lipid protons is with the methy-
lene peak [19], while in the case of the NAP-22-peptide
there is a more intense cross-peak with the terminal

methyl group of the acyl chain, particularly when cho-
lesterol is present (Fig. 5). The depth of insertion is
not greatly altered when all but two of the Phe resi-
dues of the MARCKS peptide are replaced with Ala
[19]. However, when all five Phe residues are replaced
with Ala, spin label studies indicate less penetration of
the peptide into the membrane [29]. Nevertheless, this
Ala substituted peptide has only somewhat diminished
ability to sequester PtdIns(4,5)P2. This is not that dif-
ferent from the effects of removal of the Tyr residue
from the NAP-22 peptide when studied in membranes
containing cholesterol. However for membranes devoid
of cholesterol, the Y11L-NAP-22-peptide has greater
activity in sequestering PtdIns(4,5)P2 than the unmodi-
fied NAP-22 peptide. We suggest that the ability of
peptides to form domains of PtdIns(4,5)P2 is a conse-
quence of the combined interactions of the cationic
cluster of amino acid residues and the insertion of
hydrophobic amino acids into the membrane. In some
cases, the insertion of groups that promote the forma-
tion of cholesterol-rich domains will result in the pref-
erential sequestering of PtdIns(4,5)P2 into one of the
domains. This would be a mechanism additional to the
direct electrostatic interaction between the peptide and
PtdIns(4,5)P2.
When electrostatic interactions predominate, there is
sequestering of PtdIns(4,5)P2, independently of the nat-
ure of the surrounding lipid. However, when the elec-
trostatic interactions are reduced, as it is in NAP-22
compared with the MARCKS peptide, then sequester-

ing of PtdIns(4,5)P2 is also affected by the insertion of
hydrophobic moieties into the membrane that change
the depth of burial of the peptide, the orientation of
the peptide with respect to the membrane, and the lat-
eral distribution of lipids into domains through hydro-
phobic interactions. These hydrophobic interactions
alone are insufficient in the case of the Y11L-NAP-22-
peptide to modulate the sequestering of cholesterol. In
the case of NAP-22, the combined interactions of the
myristoyl group, the Tyr side chain and the cationic
cluster in the peptide, result in a cholesterol-dependent
sequestering of PtnIns(4,5)P2 into domains.
There is also a structural aspect that makes NAP-22
unusual. Many proteins are N-terminally myristoylated
[30] but only a few have in addition, clusters of cationic
residues comprised of four or more Lys or Arg residues
in sequence. One of the few examples we have found is
the membrane fusion protein, p15, of baboon reovirus
that is both myristoylated and has a cluster of four
cationic residues [31]. Two other examples we have
discsussed earlier are MARCKS and NAP-22. The
structural difference between these two proteins is that
the cationic cluster of NAP-22 is close to the myristoyl
group at the amino terminus. This is not the case for
MARCKS. Since myristoylation is a factor that causes
proteins to sequester into raft domains, it would seem
a priori more likely that sequestering of PtnIns(4,5)P2
would be coupled to translocation to a cholesterol-rich
domain for NAP-22 than for MARCKS, as is found.
The rearrangement of PtdIns(4,5)P2 and cholesterol

in a membrane caused by the presence of NAP-22 pro-
vides a mechanism by which this protein can affect the
actin cytoskeleton. PtdIns(4,5)P2 plays an important
role in the attachment of the cytoskeleton to the
plasma membrane as well as affecting actin dynamics
[32]. Since NAP-22 causes the sequestering of both
R. F. Epand et al. Cholesterol-dependent lipid rearrangement
FEBS Journal 272 (2005) 1792–1803 ª 2005 FEBS 1799
cholesterol and PtnIns(4,5)P2 into domains, we suggest
that the protein recruits more PtnIns(4,5)P2 into raft-
like domains. This will result in an increase in the
interactions between the cytoskeleton and plasma
membrane occurring at rafts and hence the rearrange-
ment of the spatial distribution of the cytoskeleton. In
neurons, several proteins including NAP-22, GAP-43
and MARCKS, affect the efficiency of raft dependent
signaling [33]. Both the kinase that catalyses the syn-
thesis of PtdIns(4,5)P2 [34] as well as the phosphatase
that degrades it [35], affect cytoskeletal organization.
NAP-22 together with related proteins, function to
enhance the accumulation and assembly of PtdIns(4,
5)P2-rich raft domains [36]. During neuronal develop-
ment, axonal elongation and branching are regulated
by the activity of PI(4)P5 kinase [37], an enzyme that
synthesizes PtdIns(4,5)P2. Thus, the amount and distri-
bution of PtdIns(4,5)P2 will regulate cytoskeletal
dynamics, which in turn will affect neuronal growth
and development. CAP-23 accumulates in the neuronal
growth cone and has a marked effect on the rearrange-
ment of the actin cytoskeleton [38]. An early conse-

quence of CAP-23 accumulation is an increase in
dynamic actin structures and the loss of more stable
actin filaments such as stress fibers.
We can use this simplified system to identify certain
molecular interactions that we can suggest form the
basis for events that are observed at the cellular level.
In this work we use a 19 amino acid lipopeptide cor-
responding to the amino terminus of NAP-22. With
this peptide, the consequences of the rearrangements
of PtdIns(4,5)P2 we observe by fluorescence or by
DSC are significantly greater than we observe with the
intact protein. It is known that there are N-terminal
fragments of NAP-22 present in cells [2]. Furthermore,
a construct composed of the N-terminal segment of
CAP-23 and containing 40 amino acids arranges in a
punctate pattern on the cell surface and is associated
with the cytoskeleton. Like the full length protein, this
short construct produces marked changes in cell mor-
phology but unlike the full length protein, it does not
produce blebbing [38]. It has been estimated that
PtnIns(4,5)P2 comprises 0.3–1.5% of the phospholipid
of the plasma membrane of mammalian cells [12]. If
dissolved in the total cell volume, this amount of
PtnIns(4,5)P2 would have a concentration in the range
2–30 lm, although the PtnIns(4,5)P2 varies consider-
ably among cell types and is particularly low in
some cells [39]. Nevertheless, our use of 0.2 mol%
PtnIns(4,5)P2 in the model membranes is within the
physiological range. In comparison, in the developing
brain NAP-22 comprises 0.4–0.8% of the total protein,

corresponding to a concentration of 20–40 lm [40].
Thus, there are comparable amounts of PtnIns(4,5)P2
and NAP-22 in the cell and the ratio is within the
range used in our work. Since NAP-22 binds to
PtnIns(4,5)P2 by nonspecific electrostatic interactions,
one molecule of NAP-22 can promote the formation
of a domain of many molecules of PtnIns(4,5)P2 [27].
Thus not all of the NAP-22 has to be bound to
PtnIns(4,5)P2 in order for a major fraction of this
lipid to be sequestered into a domain. This is different
from proteins with specific folded domains that bind
PtnIns(4,5)P2 in a stoichiometric fashion [41]. The
greater potency of the N-terminal peptide in forming
domains would suggest that membrane lipid domain
formation may be facilitated by proteolytic processing
of NAP-22. Myristoylated proteins interacting with
membranes through both electrostatic interactions as
well as insertion of a myristoyl group, can be dissoci-
ated from the membrane by proteolytic cleavage [42].
It is possible that this is an example of the opposite,
i.e. proteolytic cleavage would cause increased seques-
tration to the membrane by removing the anionic por-
tion of the protein that would repel anionic lipids. The
pI of rat NAP-22 is only 4.5. Another indication of
the importance of the amino terminal fragment of
NAP-22 is that the first 21 amino acids are invariant
among NAP-22 of several mammalian species and
this segment differs by only one residue with chicken
NAP-22 (CAP-23).
Thus both cholesterol and PtdIns(4,5)P2 affect the

location of the NAP-22-peptide in a bilayer. The lipo-
peptide has little capability of inducing phase separ-
ation in mixtures of SOPC and cholesterol, but with
addition of PtdIns(4,5)P2 there is a cholesterol-
dependent separation into a cholesterol enriched and a
cholesterol-depleted domain. This segregation is repre-
sented in the drawing in Fig. 8 (not drawn to scale).
These results demonstrate how sensitive the interaction
Fig. 8. Schematic representation of the domain enrichment caused
by the peptide (red) in the presence of cholesterol (blue) and
PtdIns(4,5)P2 (green). The other lipid headgroups are presented in
grey and the acyl chains in orange. The clustering of charges in the
peptide permits interaction with the negative charges on the head-
group of PtdIns(4,5)P2 concomitantly resulting in the redistribution
of cholesterol.
Cholesterol-dependent lipid rearrangement R. F. Epand et al.
1800 FEBS Journal 272 (2005) 1792–1803 ª 2005 FEBS
of even small peptides with membranes is to the lipid
composition of the membrane.
Experimental procedures
Materials
The synthetic lipopeptide with the sequence: myristoyl-
GGKLSKKKKGYNVNDEKAK-amide, corresponding to
the 19 amino terminal residues of NAP-22, as well as a
variant of this lipopeptide, Y11L were purchased from Bio-
Source International (Hopkinton, MA, USA). Phospho-
lipids and cholesterol were purchased from Avanti Polar
Lipids (Alabaster, AL, USA). PtdIns(4,5)P2 was purified
from porcine brain. Bodipy-TMR-PI(4,5)P2 was purchased
from Molecular Probes (Eugene, OR, USA).

Preparation of samples for DSC and NMR
experiments
Lipid components were codissolved in chloroform ⁄ meth-
anol (2 : 1, v ⁄ v). For samples containing peptide, an ali-
quot of a solution of the peptide in methanol was added to
the lipid solution in chloroform ⁄ methanol. The amount of
peptide used was monitored by the absorbance at 280 nm
using an extinction coefficient calculated from the amino
acid composition [43]. The solvent was rapidly evaporated
at 30 °C under a stream of nitrogen with constant rotation
of a test tube to avoid separation of lipid components [12]
and to deposit a uniform film of lipid over the bottom
third of the tube. Last traces of solvent were removed by
placing the tube under high vacuum for at least 2 h. The
lipid film was then hydrated with 20 mm Pipes, 1 mm
EDTA, 150 mm NaCl with 0.002% NaN
3
, pH 7.40 and
suspended by intermittent vortexing and heating to 50 °C
over a period of 2 min under argon. Samples used for
NMR analysis were hydrated with the same buffer made in
2
H
2
O and adjusted to a pH meter reading of 7.0 (pD ¼
7.4) and incubated at least 24 h at 4 °C to allow conversion
of any anhydrous cholesterol crystals to the monohydrate
form. For the NMR measurements, the samples were first
spun in an Eppendorf centrifuge at room temperature. The
resulting hydrated pellet was transferred to a 4 mm zirco-

nia rotor with the 12-lL Kel-F insert, attempting to pack
the maximal amount of lipid into the rotor while keeping it
wet.
DSC
Measurements were made using a Nano Differential Scan-
ning Calorimeter (Calorimetry Sciences Corporation,
American Fork, UT, USA). The scan rate was 2 ° CÆmin
)1
and there was a delay of 5 min between sequential scans in
a series to allow for thermal equilibration. The features of
the design of this instrument have been described [44]. DSC
curves were analyzed by using the fitting program, DA-2,
provided by Microcal Inc. (Northampton, MA, USA) and
plotted with origin, version 5.0.
Preparation of LUV for fluorescence spectroscopy
A solution of POPC and 0.1 mol% Bodipy-TMR-PI(4,5)P2
with or without 40 mol% cholesterol was prepared in chlo-
roform ⁄ methanol (2 : 1) and the lipid deposited on the
walls of a glass test tube by solvent evaporation with a
stream of nitrogen gas. Last traces of solvent were then
removed by evaporation for 2 h under vacuum. Films were
hydrated with a 10 mm Hepes buffer pH 7.4 containing
1mm EDTA and 140 mm NaCl. The lipid suspensions were
further processed by five cycles of freezing and thawing, fol-
lowed by 10 passes through two stacked 0.1 lm polycar-
bonate filters, using a Lipex extruder [45], to convert the
lipid suspension to LUVs. The content of lipid phospho-
rous was determined by the method of Ames [46].
Fluorescence quenching
Fluorescence measurements were made in silanized glass

cuvettes containing 2 mL of the appropriate buffer, at
25 °C, under constant stirring with Teflon magnets. An
amount of LUVs were added to the cuvette and then titra-
ted with successive additions of small aliquots of peptide
solution, using silanized Eppendorf tips. Peptide solutions
were made in the appropriate buffer and the peptide con-
centration was quantified by absorbance at 275 nm. Peptide
solutions were kept in silanized containers at 4 °C until
used.
The excitation and emission monochromators were set at
542 nm and 571 nm, respectively, with a 500-nm cut-off fil-
ter in the emission path. The excitation and emission band-
pass slits were set at 4 nm. Cuvettes were maintained in the
dark with the shutters closed between additions of peptide;
the shutter was toggled only at the beginning of the record-
ing of each emission scan, to prevent photobleaching of the
probe. Two independent determinations were performed
with each batch of LUVs. The corresponding set of titra-
tion curves with buffer not containing peptide were subtrac-
ted from the titration with peptide.
1
H NOESY MAS/NMR
High resolution MAS spectra were acquired using a spin-
ning speed of 5.5 kHz in a Bruker AV 500 NMR spectro-
meter. Probe temperature was 24 ± 1 °C. The 2D NOESY
spectra were obtained using delay times of 50 and 300 ms.
Resonances were assigned based on reports of phosphat-
idylcholine [18], cholesterol [47] and amino acid residues
[48].
R. F. Epand et al. Cholesterol-dependent lipid rearrangement

FEBS Journal 272 (2005) 1792–1803 ª 2005 FEBS 1801
13
C DP MAS/NMR
The same 4-mm zirconia rotor with the 12-lL Kel-F insert
was used to acquire
13
C DP MAS ⁄ NMR in a Bruker
Avance 300 spectrometer operating at 75.48 MHz for
13
C.
The spectra were referenced to an external standard of gly-
cine crystals, assigning a chemical shift of 176.14 p.p.m. for
the carbonyl carbon. Samples were spun at 5 kHz. The
temperature inside the rotor was 25 ± 1 °C. Single pulse
excitation with high power proton decoupling was used
with a 4 lsec pulse for
13
C and the proton frequency opti-
mized for decoupling. A recycle time of 5 s was used.
Acknowledgements
This work was supported by grant MT-7654 from the
Canadian Institutes of Health Research.
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