Conformational Changes and Association of Membrane-Interacting

Nov 12, 2015 - Department of Chemistry and Biochemistry, Wilfrid Laurier University, 75 University Ave. W., Waterloo, Ontario, Canada N2L 3C5. ABSTRAC...
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Conformational Changes and Association of Membrane-Interacting Peptides in Myelin Membrane Models: A Case of the C‑Terminal Peptide of Proteolipid Protein and the Antimicrobial Peptide Melittin Ashtina Appadu, Masoud Jelokhani-Niaraki, and Lillian DeBruin* Department of Chemistry and Biochemistry, Wilfrid Laurier University, 75 University Ave. W., Waterloo, Ontario, Canada N2L 3C5 ABSTRACT: Model membranes composed of various lipid mixtures can segregate into liquid-ordered (Lo) and liquid-disordered (Ld) phases. In this study, lipid vesicles composed of mainly Lo or Ld phases as well as complex lipid systems representing the cytosolic leaflet of the myelin membrane were characterized by fluorescence resonance energy transfer with a donor/acceptor pair that preferentially partitioned into Lo or Ld phases, respectively. The fluidity of the lipid systems containing >30% cholesterol was modulated in the presence of the amphipathic peptide melittin. With all the studied lipid systems, melittin attained an α-helical conformation as determined by CD spectroscopy and attained varying degrees of membrane association and penetration as determined by intrinsic Trp fluorescence. The other protein domain utilized was a putative amphipathic helical peptide derived from the cytosolic C-terminal sequence of proteolipid protein (PLP) which is the most abundant protein in the myelin membrane. The C-terminal PLP peptide transitioned from a random coil to an α-helix in the presence of trifluoroethanol. Upon interacting with each of lipid vesicle system, the PLP peptide also folded into a helix; however, at high concentrations of the peptide with fluid lipid systems, associated helices transmuted into a β-sheet conformer. The membrane-associated aggregation of the cytosolic C-termini could be a mechanism by which the transmembrane PLP multimerizes in the myelin membrane.

1. INTRODUCTION Plasma membranes are complex bilayers of lipids and proteins that can be segregated into various regions or microdomains such as lipid rafts. Typically, lipid rafts are enriched in cholesterol, sphingomyelin, and unsaturated lipid and are less fluid or more ordered than the surround membrane environment. Proteins can be sequestered into or excluded from the lipid rafts as they coalesce into functional or signaling platforms.1−4 Lipid rafts have been detected by direct or indirect methods in plasma membranes of almost all cell types.5 The myelin sheath is a multilamellar plasma membrane that propagates from oligodendrocytes in the central nervous system (CNS) and the Schwann cells in the peripheral nervous system (PNS). Microdomains including lipid rafts have been characterized in oligodendrocytes, Schwann cells, and the myelin membrane from the CNS and PNS. 6,7 Model membranes composed of lipid mixtures can have the coexistence of liquid-ordered (Lo) and liquid-disordered (Ld) phases.8 The membrane fluidity can be modulated by the presence of cholesterol. The well-studied ternary system of 1palmitoyl-2-oleoyl-glycero-phosphocholine (POPC), sphingomyelin, and cholesterol (in equimolar amounts) is mainly a Lo phase representing the canonical lipid raft microdomain.9 The interaction of proteins with myelin membranes is essential for the physiological function of myelinated nerve fibers. To gain better understanding of the mutual interaction of myelin membranes and protein motifs, this study characterized the fluidity of various myelin-like lipid systems © 2015 American Chemical Society

in the absence and presence of a peptide representing protein amphiphatic secondary structures. Conversely, the conformational changes and association of the amphipathic protein structure upon interaction with the lipid systems were also analyzed in detail. Melittin, a well-studied antimicrobial peptide, was chosen as the prototypical amphipathic helical peptide. This 26 amino acid peptide (sequence: GIGAVLKVLTTGLPALISWIKRKRQQ-NH2) with an amidated C-terminus exists as random coil in aqueous solution but can spontaneously convert to an α-helix once in contact with lipid membranes.10,11 To expand the scope of this study, the influence of the myelinlike lipid membranes on the conformation of myelin-relevant protein structure was also investigated. A peptide segment from the C-terminus of the proteolipid protein (PLP) which is highly abundant in the CNS myelin, was synthesized for this purpose. The structure of this peptide segment (I258AATYNFAVLKLMGRGTKF277; human sequence) has not been previously characterized but is predicted to be an amphipathic α-helix. PLP is an extremely hydrophobic integral membrane protein consists of four transmembrane α-helices which are connected by extracellular and intercellular loops with the N- and Ctermini residing on the cytosolic side.12 PLP maintains a structural role in myelin compaction where the extracellular loops from opposing membranes provides strut-like support Received: July 30, 2015 Revised: October 28, 2015 Published: November 12, 2015 14821

DOI: 10.1021/acs.jpcb.5b07375 J. Phys. Chem. B 2015, 119, 14821−14830

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The Journal of Physical Chemistry B within the electron microscopy observable intraperiod line of the multilayered compact myelin. Injection of PLP into rodents and other animals induces experimental autoimmune encephalomyelitis (EAE), an inflammatory demyelinating model of multiple sclerosis. Mutations within the PLP gene are associated with Pelizaeus-Merzbacher disease and some forms of spastic paraplegia.

Table 1. Lipid Composition of the Various Membrane Model Systems Investigated membrane model

system 1 2 3

2. MATERIAL AND METHODS 2.1. Materials. Melittin (HPLC purity ≥85%) was purchased from Sigma-Aldrich (Oakville, ON, Canada) and the N-acetylated C-terminal peptide of PLP, residues 258−277 of the human sequence (Ac-IAATYNFAVLKLMGRGTKF) (HPLC purity ≥95%) was custom synthesized by AAPPTec . Both peptides were dissolved in pure Milli-Q water and their final concentrations determined using the Edelhoch method.13 1-Palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC), 1palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS), egg sphingomyelin (SM), and cholesterol were purchased from Avanti Polar Lipids, as were the fluorescent probes 1,2dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-7-nitro-2-1,3benzoxadiazol-4-yl (NBD-DPPE) and 1,2-dioleoyl-sn-glycero-3phosphoethanolamine-N-lissamine rhodamine B sulfonyl (Rhod-DOPE). 2.2. Preparation of Vesicles. All lipids were prepared as stock solutions (25 mg/mL) in chloroform. The various lipids were mixed in a round-bottom flask and dried under a mild nitrogen gas. For resonance energy transfer (RET) measurements, the fluorescent probes, NBD-DPPE and Rhod-DOPE, were also added to final concentrations of 0.8 and 0.2% mol, respectively. The resulting thin lipid film was further dried overnight in a vacuum desiccator to remove traces of residual solvent. The lipid films were hydrated in buffer solution and briefly exposed to a bath sonicator to ensure the resuspension. For all fluorescence measurements, lipids were resuspended in 5 mM HEPES 150 mM NaCl buffer at pH 7.4, whereas vesicles prepared for CD measurements were resuspended in 100 mM Tris, 10 mM NaF buffer, pH 7.4 in order to minimize absorption interferences. The lipid suspensions were then frozen and thawed five times before being passed 19 times through a 100 nm polycarbonate membrane using a syringe extruder (Avestin, Ottawa, Canada) to give uniform large unilamellar vesicles (LUVs) with a final total lipid concentration of 1 mM. The lipid composition of the various membrane model systems are listed in Table 1. For the various analyses (see below), the lipid−peptide ratio was varied by changing the peptide concentration and keeping the lipid concentration constant at 1 mM. 2.3. Fluorescence Spectroscopy. For the RET analysis, NBD-DPPE served as the donor probe and Rhod-DOPE as the acceptor probe with the final probe to total lipid ratio of 1:125 (0.8% mol) and 1:500 (0.2% mol), respectively. Steady-state fluorescence emission spectra were obtained using a Varian Eclipse spectrofluorometer maintained at 25 °C. Measurements were taken at an excitation wavelength of 428, 460, or 560 nm, while the fluorescence emission was collected between 450 and 650 nm or between 570 and 650 nm. For each lipid system investigated, measurements were completed on vesicles only (no probes), with only donor probe, with only acceptor probe, and with donor and acceptor probe. The preparations with only the donor or acceptor probe serves as controls for concentration corrections. The last preparation, containing

4 5

fluid lipid raft plasma membrane healthy myelin diseased myelin

lipid compositiona

lipid ratio (mol %)

PC PC/SM/CHOL PC/PE/PS/CHOL

100 33.3:33.3:33.3 45:25:10:20

PC/PS/PE/SM/CHOL

25.9:7.3:29.0: 6.2:31.6

PC/PS/PE/SM/CHOL

20.1:7.4:32.9:2.2:37.4

a

PC, POPC; PE, POPE; PS, POPS; CHOL, cholesterol; SM, sphingomyelin.

both donor and acceptor probes, was used to monitor energy transfer. Vesicles were allowed to equilibrate for an hour at 25 °C before measurements. For all lipid systems, the RET analyses were repeated with melittin at a final concentration of 10 μM. The RET efficiency (E) was then calculated, both in the presence and absence of melittin, to establish a fluidity profile for each of the lipid systems, using eq 1 where IDA is the intensity in the presence of both donor and acceptor and ID is the intensity of the donor only. I E = 1 − DA ID (1) The Trp fluorescence of melittin in the various lipid systems was measured from 300 to 450 nm with the excitation wavelength was set to 280 nm. All measurements were at 25 °C. 2.4. Circular Dichroism (CD) Spectroscopy. Melittin or the PLP peptide, at various concentrations, was added to the vesicles, incubated at 25 °C, and then pulse centrifuged. All measurements were completed with an Aviv 215 spectropolarimeter using a quartz optical cell with a path length of 0.1 cm at 25 °C (unless otherwise specified), and the spectra were recorded in 0.5 nm increments with a 1 s response and a bandwidth of 1 nm. An average of four scans was taken per sample and ellipticity values were converted to mean residue ellipticities (MRE). The fractional α-helical content ( fα) of melittin was estimated from the molar ellipticities at 222 nm using fα =

θ222 − θRC θH − θRC

(2)

where θ222 is the ellipticity at 222 nm and θRC and θH are the ellipticity for the fully random coil and the fully helical conformation, respectively. The previously determined values of θRC = −1500 deg cm−2 dmol−1 and θH = −33400 deg cm−2 dmol−1 were adopted.14 The CD spectra were also completed with the PLP peptide in buffer and in 20, 50, and 80% (v/v) 2,2,2,-trifluoroethanol (TFE).

3. RESULTS AND DISCUSSION In the cell, lipid microdomains of the plasma membrane are dynamic entities that are modulated during aging and in disease.15−17 In model membrane systems, lateral heterogeneity can give rise to Lo domains coexisting within the Ld phase, representing lipid rafts within a fluid membrane. Besides the fluid POPC system and the well-characterized lipid raft 14822

DOI: 10.1021/acs.jpcb.5b07375 J. Phys. Chem. B 2015, 119, 14821−14830

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The Journal of Physical Chemistry B membrane system, several lipid compositions mimicking a simplified plasma membrane and the myelin membrane were utilized in this study (see Table 1). The “healthy myelin” and “diseased myelin” (or “EAE-like myelin”) membrane systems represent the cytoplasmic leaflet of the myelin membrane as previously determined from the lipid composition of control and EAE-induced marmoset white matter, respectively.18 These complex model myelin lipid systems have been utilized in various studies with myelin basic protein.19−21 The major difference between the “healthy” and “diseased” myelin is the increase in cholesterol and decrease in sphingomyelin in the “diseased” state. Under active multiple sclerosis, there is a decrease in the sphingolipid content in human brain white matter.22 3.1. Lipid Fluidity of Various Membrane Systems. In vitro, phase separation can occur at an intermediate temperature in model membranes consisting of three components that include a low Tm lipid (such as POPC), a high Tm lipid (such as a sphingolipid or a saturated lipid), and cholesterol. RET analysis was used to characterize the phase separation in the different the model membrane systems including those mimicking the healthy and the diseased state of the myelin sheath. For this purpose, two probes, NBD-DPPE (as donor) and Rhod-DOPE (as acceptor), each known to preferentially partition in Lo and Ld phases, respectively, were selected.23−25 By monitoring donor quenching in the presence of the acceptor, quantitative measurements of the energy transfer efficiency (E) were obtained in each of the model membrane systems, both in the absence and presence of melittin. Melittin was selected as a representative protein amphipathic motif since it is a widely studied amphipathic peptide that readily interacts with various membranes. Furthermore, its single Trp residue permits fluorescence analysis to determine its degree of membrane partitioning (see section 3.2). The POPC system and the lipid raft model membrane were used to represent the two extremes in terms of the bilayer fluidity. In comparison to a pure single fluid crystalline lipid, a system with Lo domains within the Ld phase will give rise to a decrease in RET efficiency as the average donor−acceptor distance increases. The energy efficiency of each lipid systems was determined using eq 1, both in the presence and absence of melittin, and the values obtained provided an estimation of the overall fluidity of each of the model systems as well as general information about the size of the domains present in each. As expected, the RET analysis determined the highest energy efficiency with the NBD-DPPE/Rhod-DOPE pair in LUVs comprised only of POPC (system 1; see Table 1) and the lowest efficiency, approximately 55% less, in the lipid rafts (system 2). The energy efficiencies for the plasma membrane, healthy myelin, and diseased myelin model membranes (systems 3−5) were similar or slightly less than that in the POPC vesicles (Figure 1, blue bars). Cholesterol can modulate the fluidity of membranes by promoting the tight packing of saturated lipids. Moreover, the preferential interaction of cholesterol with sphingomyelin in phosphatidylcholine membranes leads to lateral heterogeneity and L o domain formation.26 In the systems examined, the extent of phase separation can be correlated with a low ratio of cholesterol to sphingomyelin, rather than with solely a high content of cholesterol. The RET efficiency observed for the POPC and plasma membrane systems are similar, and thus the plasma membrane system (20% cholesterol, no sphingomyelin) may not support Lo domain formation. Both healthy and diseased

Figure 1. Average RET efficiency (E) in the various lipid systems. The average E was determined from three trials performed for each lipid system with NBD-DPPE and Rhod-DOPE as the donor/acceptor pair. The blue bars represent the energy efficiency in the absence of the model peptide melittin whereas the red bars indicate the efficiency in the presence of 10 μM melittin. Refer to Table 1 for the composition of each lipid system.

myelin vesicles are complex in lipid composition with cholesterol/sphingomyelin ratios of 1:5 and 1:22, respectively, and may contain Lo domains due to the cholesterol− sphingomyelin association. However, the size and/or number of Lo domains may be different in these two systems in comparison to the lipid raft system. The formation of smaller Lo domains, or nanodomains, in the model myelin systems would result in a decrease in the distance between the donor and acceptor probes, thus causing an increase in energy transfer.24 Monolayers of healthy and EAE-like cytosolic myelin lipids form coexisting immiscible liquid phases.27Also, lateral heterogeneity in model membranes composed of extracted myelin lipids occurs.28 The addition of melittin to the lipid systems resulted in changes in the energy efficiencies except for the plasma membrane model and POPC vesicles, supporting that these two systems do not have any significant phase separation in the presence and absence of the peptide. The efficiencies decreased by a third in the healthy myelin and diseased myelin membrane systems in the presence of melittin whereas an increase by a third was observed in the lipid raft model system (see Figure 1, red bars). The previously estimated Lo domain radii in POPC/ SM/CHOL (1:1:1) large vesicles at 23 °C is 8−10 nm; however, the domain size increases in the presence of the detergent triton X-100 or a transmembrane peptide.29 A more recent analysis suggests the nanodomains to be 30% mol) content. As cholesterol orders its membrane environment, the interaction of planar cholesterol moiety with the Trp residue of melittin may be favored. Lastly, the spectral shape can indicate conformational heterogeneity in membrane systems.42 Asymmetry in the spectrum is noted in all systems with the greatest spectral broadening observed in lipid raft system. 3.3. The Interaction and Conformation of Melittin with the Lipid Systems. Melittin is one of the most widely studied amphiphatic membrane-lytic peptides. In a crystalline state, this 26 amino acid peptide can form a helix structure in which residues 2−11 from an α-helix followed by a kink (residues 11−13), another less defined α-helix (residues 13− 23), and a highly basic unstructured C-terminus.43 Within the helical region, a leucine zipper motif, or heptad repeat of hydrophobic Leu/Ile is found from residues 6−20.44 In aqueous solution, at low concentrations and in the absence of high salt or phosphate melittin, exists predominantly as a random coil structure. As the concentration increases, the peptide can self-associate into a dimeric structure consisting of two α-helices before finally forming tetrameric structure (dimer of dimers) at higher concentrations.45,46 A leucine zipper motif may promote the dimer association as a coiled-coil conformation of melittin.44 Also, the promotion of the random coil to helix conformation of melittin can occur upon binding to a lipid bilayer. The conformation can fluctuate slightly depending on the properties of the lipid environment including the electrostatic charge and the overall thickness of the bilayer.47−49 Melittin can bind to the surface of the bilayer, can embed partially into the membrane, or can tranverse the membrane. Similarly to many other antimicrobial peptides, melittin at higher concentrations can induce the formation of transmembrane pores into the bilayer. The formation of a torroidal pore would require the minimum of a helical tetramer. In the CD spectroscopy analysis of melittin with the various lipid vesicles (systems 1−5), the peptide concentration and buffer conditions were kept well below those promoting selfassociation and tetrameric formation in aqueous solution. In general, a spectrum with the characteristic minima at 208 and 222 nm indicating an α-helical conformation of melittin was observed with each of the lipid systems (see Figure 3). The

when the temperature is above the gel-to-liquid crystalline phase transition temperature (Tc); however, as the temperature is lowered to near the Tc, clustering of the peptide leads to phase separation and the subsequent fusion of vesicles.34 3.2. Partitioning of Melittin into Membranes. The interaction of melittin with lipid bilayers occurs initially as a surface adsorption which may be followed by a partial or full insertion into the membrane. The transmembrane orientation into lipid bilayers typically occurs with >4 mol % melittin.35 The interaction and partitioning of melittin into lipid bilayer can be monitored by the intrinsic fluorescence of the single Trp residue at position 19, since Trp fluorescence is sensitive to its dielectric environment. A blue shift in the emission maximum to varying degrees was observed with the binding and partitioning of melittin into all of the membrane systems in comparison to melittin in buffer (Figure 2). The largest blue

Figure 2. Tryptophan fluorescence of melittin in buffer and in the various lipid systems. Melittin (10 μM) was added to each lipid system and equilibrate from 5 min before measurement. The excitation wavelength was set to 280 nm. Refer to Table 1 for the composition of each lipid system.

shift (λmax ∼ 337−338 nm) was observed in the myelin-like vesicle systems followed by the plasma membrane system (λmax ∼ 339 nm). In these systems, all of which contain the negatively charged lipid POPS, the Trp residue of melittin is the least solvent exposed and can reside partially buried in the interfacial region of the bilayers. Only a minor blue shift was observed for the lipid raft system (λmax ∼ 351 nm) suggesting the peptide has minimal penetration into this predominantly Lo membrane. Melittin has a reduced affinity for membranes containing 30% or more cholesterol. It has been estimated that ∼40% of melittin is bound to POPC/CHOL (70:30) LUVs in comparison to 100% bound in POPC and POPC/POPG (97:3 and 85:15) with a lipid/peptide ratio of 90.36 The membrane fluidity due to the difference in the lipid acyl chain length and saturation modulates the Trp environment and the penetration of the peptide into the membrane. Several studies have determined that the Trp residue of melittin can penetrate into the interfacial lipid region to a depth of ∼10.5 Å from the center of the bilayer in zwitterionic and in zwitterionic/anionic lipid vesicles; however, in the zwitterionic vesicles Trp is in a region of increased water penetration. In contrast, the 14824

DOI: 10.1021/acs.jpcb.5b07375 J. Phys. Chem. B 2015, 119, 14821−14830

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Figure 3. Helical nature of melittin in the various lipid systems. CD spectra of melittin in the POPC (A), plasma membrane (B), lipid raft (C), healthy myelin (D), and diseased myelin (E) model systems at 25 °C. The fractional helical content of melittin was calculated from the spectra with the peptide at a 10 μM concentration (F). Refer to Table 1 for the composition of each lipid system.

melittin concentration-dependent spectra were unique for each of the lipid systems. The fractional α-helical content of mellitin in each of the lipid systems was calculated at the peptide concentration in which the θ222nm/θ208nm ratio (or the R2 ratio representing θn−π/θπ−π*) was approximately equal to one (see Figure 3F). With the fluid POPC bilayer, the peptide becomes predominantly α-helical in structure. As the peptide concentration increased, its interaction with the lipid bilayer increased, as reflected by the concentration-dependent depth of molar ellipticities of the minima (see Figure 3A). Interestingly, the emergence of an isodichroic point at around 217 nm for the 10, 20, and 40 μM mellitin CD spectra, could also indicate that at higher concentrations, melittin could exist as a mixture of different conformers, possibly the monomeric α-helix with dimer (as a coiled coil structure). At the higher melittin concentrations the CD spectra have ellipticity values at 222 nm that are greater than 208 nm which is typical for coiled-coil formation of α-helices.50,51 Possible coiled-coil conformers of melittin may exist at specific lipid/peptide ratios in all the studied lipid system. Helical conformations of melittin are also formed upon interaction with the plasma membrane and the myelin systems (see Figure 3B, D, and E). Also with these three systems, the intensity of the negative ellipticity indicates a high degree of melittin interaction with the bilayer. All three lipid systems contain the negatively charged lipid POPS at similar mol %, thus again indicating the importance of the electrostatic interaction of melittin at the membrane surface. With the myelin-like membranes (systems 4 and 5) and at the highest melittin concentration (40 μM), a loss of CD spectra integrity and intensity was observed, possibly due to the aggregation of vesicles. These vesicles have both negatively charged lipids and a high cholesterol content (>30%).

As for the lipid raft system, the melittin interaction is more limited as indicated by the low molar ellipticities values (Figure 3C). At the lowest concentration of melittin the CD spectrum is weak but indicates an α-helix conformation. At the higher concentrations, the peptide exists as a mix of random coil and α-helix. A significant reduction in helical content of melittin has been also observed in DOPC and POPC vesicles with the introduction of 40% cholesterol.32 The highest fractional helical content of melittin was determined in the plasma membrane lipid system which has negatively charged lipids with a low cholesterol level; the lowest helical content was calculated in the lipid raft system which is mostly Lo with a high cholesterol level (see Figure 3F). An intermediate helical content was observed in the fluid POPC system and in the myelin-like systems. The inclusion of negatively charged lipids in the vesicles is important for the membrane-induced structure of melittin when cholesterol is also present. 3.4. The Environment-Dependent Conformation of the C-Terminal PLP Peptide. To investigate the effect of the various model lipid membrane systems on a CNS relevant protein, the conformational properties of a segment of the Cterminus of the myelin-specific protein, PLP, was analyzed by CD spectroscopy. By analyzing the sequences of the putative cytoplasmic loop regions of PLP using various secondary structure and helical prediction programs, it was found that the C-terminus region 258−277 (human sequence) has a helical propensity and may represent an amphipathic α-helix or a 310helix (see Figure 4). The 310-helix has been well characterized in proteins and in synthetic peptides containing α,αdisubstituted amino acids.52,53 In aqueous media, the 310-helical structure gives rise to a CD spectrum similar to an α-helix conformer with a slight blue shift in the double minima (∼222−220 and ∼208−205 nm) and a reduced intensity of the π → π* transition with R2 ratio values typically ∼0.4.54 However, depending on the total net helicity this value can 14825

DOI: 10.1021/acs.jpcb.5b07375 J. Phys. Chem. B 2015, 119, 14821−14830

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the various TFE solutions, relatively ellipticities were exhibited in the lipid systems with the exception of POPC (see below). At 20 μM peptide with complex lipid systems representing lipid rafts, healthy myelin, and diseased myelin, the spectra did have a double minima, with the greatest negative ellipticity at ∼202− 204 nm indicating that the peptide was mainly in random coil with some α-helix contribution (Figure 5B). With the POPC vesicles, the spectrum of the PLP peptide at low concentration (10 μM) was representative of an α-helix (Figure 5C); however, the peptide is converted to another conformation(s) at higher concentrations. At the highest concentration (60 μM) of the PLP peptide with POPC system, there was noticeable turbidity of the solution, most likely due to aggregation of the vesicles. This resulted in loss of all spectral features. In past studies the full-length PLP was found to induce the aggregation of various lipid vesicles including those with negatively charged lipids or cholesterol.62,63 With the above-mentioned complex lipid systems (#3−5) in the presence of 60 μM peptide and with the plasma membrane system and 40 μM peptide, the CD spectra also represented a mainly helical conformation (Figure 5C). The double minima were at ∼205−207 nm and at ∼220− 221 nm. The R2 ratios ranged from 0.81 to 0.91 and are similar to those of the α-helix conformation of the peptide in TFE solutions. The CD spectra of 310-helices can be difficult to distinguish from those of mixtures of random coil and α-helix conformations.55 The last group of spectra (Figure 5D) are representative of a helix to beta conformer transformation. The prototypical CD spectrum of a β-sheet protein/peptide is characterized with a positive ellipticity maximum at 195 nm and a single negative minimum at 215 nm. The observed spectra may be the additive effects of β-sheet and helix contributions, since the first minimum is ∼206 nm and the second minimum is ∼218 nm, and the second minimum has a much greater negative molar ellipticity than the first minimum. In this set, the PLP concentration is high and temperature is an important factor. With the POPC vesicles, the PLP concentration is 40 μM, whereas with the other systems (plasma membrane, healthy myelin, and diseased myelin) the concentration is 60 μM. Also the CD spectra for the two myelin systems were measured at 37 °C. Recall that with these two myelin-like lipid systems at 25 °C and 60 μM PLP peptide (Figure 5B), the CD spectra were indicative of a helical conformation. Thus, the increase in temperature with the high PLP peptide concentration promoted the helix to beta transition in the myelin-like lipid systems whereas with POPC and the plasma membrane systems, only the higher PLP concentration was necessary to promote the transition. At 37 °C and with 60 μM PLP, the

Figure 4. C-terminal PLP peptide. The amino acid sequence and secondary structure predictions from PSIRED and YAPSIN (A). The helical wheels are illustrated on the basis of the dihehral angles of an ideal α-helix (B) and 310-helix (C) and generated using HELIQUEST.

increase to near unity.55 A dynamic equilibrium may exist between the 310- and α-helix, and the 310-helix may be part of the random coil ↔ α-helix folding/unfolding pathway.56−59 The peptide in aqueous TFE solutions exhibited CD spectra typical of a monomeric α-helix with minima at 208 and 222 nm of similar intensity of molar ellipicities (see Figure 5). Even at a relatively low concentration of TFE (20%) in buffer, the PLP peptide adopts an α-helical conformation. In buffer only, the spectrum of the peptide with a minimum at 202 nm indicates a predominantly random coil with some helical structure contribution. The R2 ratio from spectra of the PLP peptide at concentrations ranging from 10 to 60 μM in 20%, 50%, and 80% TFE ranged from 0.82 to 0.95 which is typical for the αhelix conformation. There was no indication of 310-helix formation; however, with other various C-terminal peptides, the presence of TFE in the aqueous buffer has promoted the conversion of 310-helices to α-helices.60 Furthermore, the environment of the peptide can favor one helix conformation over the other and the 310-helical conformation can be stabilized by the membrane bilayer.61 Conformational change of the PLP peptide occurred as it interacted with the bilayers of the various lipid systems. Overall, the secondary structure of the peptide was highly dependent on the lipid system and its fluidity as well as the peptide concentration. The spectra were divided into three groups representing (1) admixtures of α-helix and random coil conformations (or short helix within the peptide (Figure 5B), (2) mainly α-helix (Figure 5C), and (3) β-sheet formation with still some contribution from α-helix (Figure 5D). Although strong ellipticities were observed with 10 μM PLP peptide in

Figure 5. CD spectra of the PLP C-terminal peptide. The peptide (10 μM) is converted from random coil to an α-helical structure in TFE solutions (A). In the complex lipid systems, the peptide (20 μM) is mostly random coil with some helical content (B). The peptide attains a helical conformation in all lipid systems at 10 μM (in POPC), 40 μM (in plasma membrane model system), and 60 μM (lipid raft and myelin lipid systems) (C) concentrations. At high peptide concentration and with temperature dependence, the PLP peptide transitions from α-helix to β-sheet (D). 14826

DOI: 10.1021/acs.jpcb.5b07375 J. Phys. Chem. B 2015, 119, 14821−14830

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membrane in healthy and diseased states significantly changes the RET efficiencies as measured with the donor: accept pair of NBD-DPPE and Rhod-DOPE. With the addition of the amphipathic peptide, the lipid raft system became more fluid or the Lo domains became smaller, whereas the myelin model systems became less fluid or the nanodomains coalesced. Melittin has a high affinity for the POPC and plasma membrane lipid vesicles and attains an α-helical conformation at all lipid-peptide ratios examined within these systems. With the complex lipid systems containing high cholesterol levels, melittin had a lower affinity for these membranes and exists as a partially folded species. This prototypic amphipathic peptide has one Trp residue near the unstructured basic C-terminal end. The greatest blue shift in the Trp fluorescence of melittin was observed with the lipids systems containing negatively charged lipids suggesting its deepest association with the bilayer. The amphipathic PLP C-terminus peptide revealed a flexible conformation in interacting with myelin-like and other lipid membrane model systems. Furthermore, this adaptive conformation of the peptide was found to be dependent on both its environment and concentration (see Figure 6). The peptide is

ellipticity induced by the diseased myelin system is significantly greater than that induced by the healthy myelin system suggesting a stronger interaction of the peptide with the lipid membrane as the helix to sheet transition is promoted. The CD spectra with the diseased myelin model and the POPC are very similar to maximum at 196 nm and with double minima at 206 and 218 nm, and thus both systems may have a similar degree of helix to sheet conformational change. At a lower lipid/ peptide ratio (or effectively, a high PLP peptide concentration), it is possible for a helix-to-sheet transition, since peptide− peptide interaction on the bilayer must occur. As β-sheet conformations arise on the membrane surface, possible aggregation of the vesicles can also result. The α-helix to β-sheet transition has been characterized in amyloidosis with prion protein and Alzheimer’s Aβ peptide and in various coiled-coil proteins such as those found in wool, hair, and fibrin clots. This helix-to-sheet transformation has also been observed with membrane-interacting peptides and cell penetrating peptides. The transmembrane peptides of SNARE proteins, VAMP1 and syntaxin 1, are capable of switching from α-helix to β-sheet conformations as a function of the lipid/ peptide ratio.64 The surface interaction of penetratin, a cell penetrating peptide, with various lipid vesicles and its subsequent helix to beta transition is a function of peptide concentration, lipid acyl chain unsaturation and length, and presence of negatively charged lipids.65−67 Unlike penetratin, the conformational transition of the PLP peptide is not dependent on the content of negatively charged lipids. In our study, the fluidity of the membrane and the peptide concentration promotes the helix to beta transition. At the bilayer surface, PLP binds to the lipids and the peptide attains a helical conformation. As more PLP peptide molecules bind, the helices can interact, possibly forming dimers or high order multimers. If the membrane has a certain degree of fluidity as that found in POPC or in the complex lipid systems at higher temperature, the associated helices are transformed into β-sheets. The mechanisms for the helix-to-sheet transition have not been well characterized. For amyloid formation, monomeric αhelices may form dimers or 4-helix bundles which elongate into the 310-helices before the transformation into the beta structure.68 Another possible pathway is the unfolding of associated α-helices to random coils which then refold into an antiparallel β-sheet.69 The role of PLP in oligodendrocytes and the myelin sheath is multifunctional. In the plasma membrane, PLP may exist as monomers, dimers, and trimers, and this oligomerization occurs after PLP is transported from the endoplasmic reticulum to the cell surface.70 Furthermore, PLP is distributed into lipid rafts and nonraft membrane regions or fluid domains.71−74 The current data supports the hypothesis that the multimerization of PLP molecules is driven by the fluidity of the membrane domain and the association of the cytosolic C-terminus tails of the PLP molecules as they form a β-structure on the inner leaflet of the membrane. It is also noted that transmembrane helix peptide segments of the tetraspanin PLP readily selfassociates into varying oligomeric states.75

Figure 6. Proposed adaptive conformational change of the PLP Cterminal peptide.

mainly random coil in aqueous solution but can fold into an αhelix with the inclusion of less polar TFE. The conformation transition to a helical structure was also observed in the presence of lipid vesicles. Most interestingly, at high peptide concentrations in fluid lipid systems, the PLP peptide may selfassociate and transition into a β-sheet structure. Although it is difficult to distinguish between the α-helix and 310-helix structures by CD spectroscopy, the PLP peptide could represent a putative amphipathic 310-helix. The 310-helix can be an intermediate in the random coil ↔ α-helix pathway and in the α-helix ↔ β-sheet transition. The lipid-dependent conformation flexibility can have important implication in understanding the PLP-membrane interactions. These adaptive conformations of the cytosolic PLP C-terminal peptide segment with membrane lipids may be involved in the trafficking, targeting, and sequestering of PLP to the myelin membrane, the oligomerization of PLP, and the distribution of the protein into various microdomains.



4. CONCLUSIONS Both melittin and the PLP peptide have basic residues near their C-terminus and can form amphipathic helices that interact with lipid membranes. The addition of melittin to lipid vesicles representing lipid rafts and the inner leaflet of the myelin

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The authors declare no competing financial interest.



ACKNOWLEDGMENTS This work was supported by funding from the Natural Sciences and Engineering Research Council of Canada (NSERC; Grants DG250119 (M.J.-N.) and DG357103 (L.D.)) and Canada Foundation for Innovation (CFI; grant 6786 (M.J.-N.)).



ABBREVIATIONS



REFERENCES

CNS, central nervous system; CD, circular dichroism spectroscopy; EAE, experimental autoimmune encephalomyelitis; RET, resonance energy transfer; Lo, liquid-ordered; Ld, liquiddisordered; NBD-DPPE, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-7-nitro-2-1,3- benzoxadiazol-4-yl; PLP, proteolipid protein; PNS, peripheral nervous system; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine; POPE, 1-palmitoyl-2-oleoyl-sn-glycero-phosphoethanolamine; POPG, 1palmitoyl-2-oleoyl-sn-glycero-phosphogycerol; POPS, 1-palmitoyl-2-oleoyl-sn-glycero-phosphoserine; Rhod-DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lissamine rhodamine B sulfonyl; SM, sphingomyelin; TFE, 2,2,2-trifluoroethanol; Tm, melting temperature

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