Induction of raft-like domains by a myristoylated NAP-22 peptide and its Tyr mutant


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


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-dependent 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 formation 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 cholesterol-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 1H 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.


calorimetric enthalpy


BODIPY TMR-X C6-phosphatidylinositol 4,5-diphosphate


cortical cytoskeleton-associated protein (a protein expressed in chicken having a high degree of homology to NAP-22)


direct polarization


differential scanning calorimetry


large unilamellar vesicle


magic angle spinning

NAP-22 peptide

the myristoylated amino terminal 19 amino acids of NAP-22 (myristoyl-GGKLSKKKKGYNVNDEKAK-amide)


neuronal axonal membrane protein, also referred to as brain acid soluble protein 1 (BASP1 protein), a 22 kDa myristoylated protein






l-α-phosphatidylinositol-4,5-bisphosphate from porcine brain




transition temperature.

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 probably 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 membranes [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 microscopy 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]. 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 demonstrated 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 internal reflectance fluorescence microscopy, we have shown that the sequestering of Bodipy-labeled PtdIns(4,5)P2 into domains can be a cholesterol-dependent phenomenon [13]. This was demonstrated using a myristoylated N-terminal peptide of NAP-22, Myristoyl-GGKLSKKKKGYNVNDEKAK-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 myristoylated 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 formation 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 contain 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].


Differential scanning calorimetry (DSC)

We determined the phase transitions of SOPC and mixtures of this lipid with one or more of the following components: cholesterol, PtdIns(4,5)P2 and NAP-22-peptide, using differential scanning calorimetry (DSC). For each sample, six consecutive DSC scans were run, three heating scans and three cooling scans at a scan rate of 2 °C·min−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 CH2 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 lowered 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 cholesterol has been depleted from a domain of the membrane 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 difference between the heating and cooling curves is characteristic of this transition and is caused by the slow rate of interconversion of two forms of anhydrous cholesterol crystals [15]. The polymorphic transition of anhydrous 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/cholesterol (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 cholesterol crystals have an enthalpy of 910 cal·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 crystals 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].

Figure 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-peptide 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°·min−1.

Table 1.  DSC Transition of SOPC. Transitions observed in cooling scans at 2°·min−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 componentsTm (°C)ΔH (kcal·mol−1)
-+NAP-22 peptide1.74
-NAP-22 peptide1.73.2
++NoneBroad. Transition 
+NAP-22 peptide0.70.36
++NAP-22 peptide1.60.85
+Y11L mutantNo transition. observed 
++Y11L mutant0.80.37
Figure 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°·min−1.

Table 2.  DSC transition of anhydrous cholesterol crystallites. Transitions observed in heating scans at 2°·min−1 of SOPC with 40 or 50 mol% cholesterol, as well as with added peptide.
% CholesterolPeptideΔH (cal·mol cholesterol−1)
4010% NAP-22 peptide 20
4020% NAP-22 peptide 33
4010% Y11L mutant peptide  0
4020% Y11L mutant peptide 12
5010% NAP-22 peptide 74
5020% NAP-22 peptide130
5010% Y11L mutant peptide 97
5020% Y11L mutant peptide110

Fluorescence quenching

Addition of the Y11L-NAP-22-peptide to large unilamellar 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-peptide 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.

Figure 3.

Quenching of the fluorescence emission by the NAP-22-peptide (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 (□) or Y11L-NAP-22-peptide (•). POPC with 40 mol% cholesterol with added NAP-peptide (bsl00084) or Y11L-NAP-22-peptide (bsl00072). Maximum emission intensity at 571 nm is plotted against the peptide to lipid molar ratio (P/L). LUVs were present in the cuvette at a concentration of 50 µm and the Bodipy-labelled lipids were present as 0.1 mol percentage of the total lipid.


The 1H 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 aromatic 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 summarized in Table 3. Each of the aromatic peaks was split into a doublet with a vicinal coupling constant of 7.5 Hz.

Figure 4.

1-D 1H MAS/NMR spectra of several lipid mixture (as indicated on the right of each spectrum) and also containing 10 mol% NAP-22-peptide. PC, POPC. See Table 3 for assignments.

Table 3.  Assignment of 1H NMR resonances.
AssignmentaChemical Shift (p.p.m.)
  • a

    Groups correspond to POPC, except for HDO that are the residual protons of the water and the phenolic CH of the Tyr aromatic protons from the NAP-22-peptide.

Meta phenolic CH7.2
Ortho phenolic CH6.9
Glycerol C25.4
Glycerol C34.5
Choline α4.4
Glycerol C14.1
Choline β3.7
Quaternary CH33.3

Static 31P NMR powder patterns demonstrated that all of the samples used for magic angle spinning (MAS) studies were in bilayer arrangement (not shown). Two-dimensional 1H 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 cholesterol 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. Slices 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 protons. 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 CH3 group (at 1 p.p.m.) relative to that of the CH2 resonances at 1.4 p.p.m. is larger in the presence than in the absence of cholesterol (Fig. 5).

Figure 5.

1D slices from the MAS 1H NOESY spectrum at the chemical shifts of the aromatic 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.

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 subtraction the two spectra were adjusted for small differences 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 positions 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 difference spectra peaks represent a small fraction of the original peak and may arise from imperfect alignment of the two spectra. In most cases, these resonance 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 CH2 groups, compared to the samples with cholesterol, and an increase of the peak intensity at the resonance position 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 indicates 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 presence of PtdIns(4,5)P2, but not cholesterol. It should be pointed out, however, that there could be contributions 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.

Figure 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.

Figure 7.

Same as Fig. 6 but for data with 300 ms delay time.

Peptide-induced changes in the chemical shift of the carbon atoms as measured by 13C direct polarization (DP)/MAS indicate that the peptide affects the chemical 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 13C resonances [20].

Table 4.  Peptide-induced 13C chemical shift differences of lipid resonances. Data show the chemical shift differences in p.p.m. for the indicated 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.
AssignmentChemical shift
(1 : 1)
(1 : 1) + PtdIns(4,5)P2
Acyl C = O1740.070.03− 0.10− 0.07
Acyl C = C130.00.070.04− 0.07− 0.05
Acyl C = C129.
Glycerol C2 710.040.00− 0.15− 0.11
Choline β 670.040.04− 0.02− 0.02
Glycerol C3 640.080.100.05ND
Glycerol C1 630.050.000.00ND
Choline α 600.060.03− 0.01− 0.04
Cholesterol C14/17 57− 0.030.00
Quaternary CH3− 0.04− 0.03
Cholesterol C9 51ND0.03
Cholesterol C13/C4 43− 0.03− 0.01
Cholesterol C10 370.020.00
Acyl C2 350.060.05− 0.060.01
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− 0.02
Cholesterol C18 13− 0.14− 0.15


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-terminal myristoyl group and the phenolic side chain of the Tyr residue, both of which are hydrophobic moieties 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 membranes [23]. In addition, the protein has no hydrophobic 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 biological membranes. We suggest that this group contributes 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 anhydrous cholesterol crystals than the Y11L mutant (Table 2). We suggest that these crystals form because cholesterol surpasses its solubility limit in the membrane 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 peptide. 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 completely eliminated. Tyr is an essential element in the CRAC motif, suggested to be responsible for cholesterol 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 contributing factor for cholesterol interaction. We have previously shown that the aromatic side chains of the 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 cholesterol clusters than is the NAP-22-peptide. This is indicated 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 addition, there is no evidence for the formation of a cholesterol-depleted phase with the Y11L-NAP-22-peptide, which would result in a more cooperative chain melting transition of SOPC with higher enthalpy (Table 1 and Fig. 1).

Peptides with cationic clusters, even simple oligomers 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 cholesterol 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 residue 86, far removed from the amino-terminal myristoyl 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 methylene 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 cholesterol is present (Fig. 5). The depth of insertion is not greatly altered when all but two of the Phe residues 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 different 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 unmodified NAP-22 peptide. We suggest that the ability of peptides to form domains of PtdIns(4,5)P2 is a consequence 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 formation of cholesterol-rich domains will result in the preferential 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 nature of the surrounding lipid. However, when the electrostatic interactions are reduced, as it is in NAP-22 compared with the MARCKS peptide, then sequestering 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 lateral distribution of lipids into domains through hydrophobic 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 provides 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 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 rearrangement 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 synthesis 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 development, 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 distribution 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 rearrangement of the actin cytoskeleton [38]. An early consequence 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 corresponding 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 morphology 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 µm, although the PtnIns(4,5)P2 varies considerably 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 µm[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 dissociated 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 sequestration to the membrane by removing the anionic portion 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 lipopeptide has little capability of inducing phase separation 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 represented in the drawing in Fig. 8 (not drawn to scale). These results demonstrate how sensitive the interaction of even small peptides with membranes is to the lipid composition of the membrane.

Figure 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 headgroup of PtdIns(4,5)P2 concomitantly resulting in the redistribution of cholesterol.

Experimental procedures


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 BioSource International (Hopkinton, MA, USA). Phospholipids 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/methanol (2 : 1, v/v). For samples containing peptide, an aliquot 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% NaN3, 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 2H2O and adjusted to a pH meter reading of 7.0 (pD = 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 zirconia rotor with the 12-µL Kel-F insert, attempting to pack the maximal amount of lipid into the rotor while keeping it wet.


Measurements were made using a Nano Differential Scanning Calorimeter (Calorimetry Sciences Corporation, American Fork, UT, USA). The scan rate was 2 °C·min−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 chloroform/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 1 mm EDTA and 140 mm NaCl. The lipid suspensions were further processed by five cycles of freezing and thawing, followed by 10 passes through two stacked 0.1 µm polycarbonate filters, using a Lipex extruder [45], to convert the lipid suspension to LUVs. The content of lipid phosphorous 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 titrated with successive additions of small aliquots of peptide solution, using silanized Eppendorf tips. Peptide solutions were made in the appropriate buffer and the peptide concentration 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 filter in the emission path. The excitation and emission bandpass 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 recording of each emission scan, to prevent photobleaching of the probe. Two independent determinations were performed with each batch of LUVs. The corresponding set of titration curves with buffer not containing peptide were subtracted from the titration with peptide.


High resolution MAS spectra were acquired using a spinning speed of 5.5 kHz in a Bruker AV 500 NMR spectrometer. 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 phosphatidylcholine [18], cholesterol [47] and amino acid residues [48].


The same 4-mm zirconia rotor with the 12-µL Kel-F insert was used to acquire 13C DP MAS/NMR in a Bruker Avance 300 spectrometer operating at 75.48 MHz for 13C. The spectra were referenced to an external standard of glycine 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 µsec pulse for 13C and the proton frequency optimized for decoupling. A recycle time of 5 s was used.


This work was supported by grant MT-7654 from the Canadian Institutes of Health Research.