DSpace at VNU: In Situ Investigation of Peptide-Lipid Interaction Between PAP(248-286) and Model Cell Membranes

J Membrane Biol DOI 10.1007/s00232-016-9878-1 In Situ Investigation of Peptide–Lipid Interaction Between PAP248–286 and Model Cell Membranes Khoi Tan Nguyen1,2 Received: 14 September 2015 / Accepted: February 2016 Ó Springer Science+Business Media New York 2016 Abstract Sum frequency generation vibrational spectroscopy (SFG) was utilized to investigate the interaction between PAP248–286 and the two lipid bilayer systems The present study also provides spectroscopic evidence to confirm that, although PAP248–286 is unable to penetrate into the hydrophobic core of the lipid bilayers, it is capable of interacting more intimately with the fluid-phase POPG/ POPC than with the gel-phase DPPG/DPPC lipid bilayer The helical structure content of lipid-bound PAP248–286 was also observed to be high, in contrast to the results previously reported using nuclear magnetic resonance (NMR) Collectively, our SFG data suggest that lipidbound PAP248–286 actually resembles its structure in 50 % 2,2,2-trifluoroethanol better than the structure when the peptide binds to SDS micelles This present study questions the use of SDS micelles as the model membrane for NMR studies of PAP248–286 due to its protein denaturing activity Keywords Gel-phase and fluid-phase model lipid bilayers Á PAP248–286 Á Peptide conformation Á SDS micelles Electronic supplementary material The online version of this article (doi:10.1007/s00232-016-9878-1) contains supplementary material, which is available to authorized users & Khoi Tan Nguyen k.nguyen9@uq.edu.au School of Chemical Engineering, The University of Queensland, Brisbane, QLD 4072, Australia School of Biotechnology, International University, Vietnam National University, Ho Chi Minh City, Vietnam Introduction The entry of the human immunodeficiency virus (HIV) into the host cell is believed to be increased 4–5 orders of magnitude by amyloid fibrils contained in semen, specifically, semen-derived enhancer of viral infection (SEVI) (Munch et al 2007; Rusert et al 2004) These amyloid fibrils allow HIV, which would normally be considered a weak pathogen, to easily enter the host cells and hence facilitate the AIDS pandemic that has killed 40 million people since it was first clinically observed in 1981 It has recently been found that SEVI fibrils form by self-assembly of the peptide PAP248–286, a proteolytic cleavage product of prostatic acid phosphate protein abundantly found in semen.(Arnold et al 2012; Munch et al 2007; Roan et al 2011) To shed light on the mechanism of this viral entry enhancement of these amyloid fibrils, an extensive amount of research has been done using nuclear magnetic resonance, circular dichroism, differential scanning calorimetry and transmission electron microscopy (Brender et al 2009, Brender et al 2011; Easterhoff et al 2011; Nanga et al 2009; Olsen et al 2012) However, the mechanism of the fibril formation remains poorly understood due to its inherited structural complexity as well as the hard to achieve physiological conditions of the phenomenon In the current study, sum frequency generation vibrational spectroscopy (SFG) was used to investigate the interaction between PAP248–286 and model cell membranes at very low peptide concentrations within the range of 0.2–1.0 lM As an intrinsically surface sensitive technique with superior sensitivity, SFG has been utilized extensively in studies of protein–lipid interactions in the last decade (Mauri et al 2014; Nguyen 2015; Volkov and Bonn 2013; Weidnerw and Castner 2013; Yan et al 2014; Ye et al 123 K T Nguyen: In Situ Investigation of Peptide–Lipid Interaction Between PAP248–286… 2014; Zhang et al 2014) It has been generally known that PAP248–286 is not toxic to the cell due to its inability to penetrate into the hydrophobic core of the lipid The molecular conformation of PAP248–286 (in 50 % TFE as well as the SDS micelle-bound form) has been solved by solution state NMR (Brender et al 2011; Nanga et al 2009) The helical content of PAP248–286 in 50 % TFE was observed to be 57 %, significantly higher than its SDS-bound form (30 %) (Brender et al 2009) In both environments, the helical content of PAP248–286 is divided into two helical segments which are almost perpendicular to each other in the case of 50 % TFE (Fig 1, PDB 2L77) In addition, the 3-10 helical component which was suggested to play a role in the fibril formation of the peptide (Nanga et al 2009) does not seem to exist in the NMR structure of the peptide We believe that the discrepancies between the structural properties proposed by these NMR measurements originate from the possible protein denaturing activity of SDS (Seddon et al 2004; Warschawski et al 2011), making this surfactant an inappropriate model membrane system at least for studies of interactions between cell membranes and PAP248–286 While SDS micelles have been reported to be an excellent model membrane system for studies of robust transmembrane peptides/proteins (Tulumello and Deber 2009), it has been reported to denature some membrane proteins/peptides, especially when these structures not penetrate into the hydrophobic core of the micelles (Seddon et al 2004; Warschawski et al 2011) Because the amyloidogenic activity of peptides is directly dictated by their interactions with the surrounding media, it is important to choose a model membrane system for studies of PAP248–286 that does not alter the structural property of the peptide To overcome the possibility of protein denaturation by SDS surfactant, phospholipid mixtures were used to make lipid bilayers mimicking model membranes in our study Experimental Section Materials The PAP248–286 peptide ([95 % purity) was purchased from Biomatik (Toronto, ON) Phospholipids POPC (1-palmitoyl2-oleoyl-sn-glycero-3-phosphocholine), POPG (1-palmitoyl-2-sn-glycero-3-phospho-(10 -rac-glycerol)), hydrogenated and deuterated DPPG/DPPC (1,2-dipalmitoyl (D62)-sn-glycero-3-phospho-glycerol/choline) were purchased from Avanti Polar Lipids Inc (Alabaster, AL) Freshly purified water (Ultrapure Milli-Q unit from Millipore, USA) with a resistivity of 18.2 MX cm was used to make all the solutions in the experiments These lipid species chosen in our study have been commonly used in studies of the interactions between the lipid and peptides/ proteins Deposition of the lipid bilayers on to CaF2 prisms were prepared by sequentially depositing the distal and proximal layers using a 622 Nima LB trough (Chen et al 2007) In brief, the CaF2 prism was immersed in the water trough; then a lipid monolayer at 34 mN/m surface pressure was spread on the water surface This surface pressure was maintained, while the prism was being lifted out of the subphase at the rate of mm/min The second lipid leaflet deposition was made by bringing the first deposited lipid layer into contact with a lipid monolayer at 34 mN/m surface pressure Once the lipid bilayer was formed, it was kept hydrated until the completion of the experiment The protein reservoir with a volume of ml was placed below the lipid bilayer deposited CaF2 prisms during the SFG measurement The peptide solution was stirred by a magnetic micro-stirrer at a rate of 40 rpm during the peptide– lipid interaction The pH of the bulk was stabilized with 20 mM phosphate buffer saline (PBS) at 7.2 containing 20 mM NaCl All experiments were carried out at room temperature (*23 °C) The SFG spectra in the amide I frequency region were collected from the lipid-bound Fig Two structures of PAP248–286 in SDS micelles (PDB = 2L3H, right) and 50 % TFE (PDB = 2L77, left) Dynamic α/3-10 helix 123 K T Nguyen: In Situ Investigation of Peptide–Lipid Interaction Between PAP248–286… PAP248–286 after the peptide solution had been in contact with the lipid bilayers for at least h (to equilibrate and ensure that no further time-dependent changes occurred) quantity Lxx ðxÞ in (3) will be close to zero since the angle of the transmitted beam, ht ; will approach 90 We thus have 2ị 2ị ẳ Lzz ðxÞLzz ðx1 ÞLzz ðx2 Þ sin b sin b1 sin b2 vzzz vppp ð4Þ SFG Setup In the SFG experiments, the visible and the tunable IR beams were spatially and temporally overlapped on the solution interface The visible beam was generated by frequency-doubling the fundamental output pulses (1064 nm, 10 Hz) of 36 ps pulse-width from an EKSPLA solid state Nd:YAG laser (PL2241) The tunable IR beam was generated by an EKSPLA optical parametric generation/amplification and difference frequency system based on LBO and AgGaS2 crystals Fluctuations in the beam energies were only % standard deviation in the tunable IR beam and 1.5 % in the visible beam In the current SFG measurements, the incident angle for visible beam was avis = 60° and for the IR beam it was aIR = 54° The neartotal-internal reflection experimental geometry was adopted to collect the SFG signals from interfacial PAP248–286 using a right angle CaF2 prism as the solid substrate In this study, each presented data point was averaged over 100 acquisitions ð2Þ The quantities vssp (s polarized SFG, s polarized visible ð2Þ and p polarized infrared polarization combination) and vppp (p polarized SFG, p polarized visible and p polarized infrared polarization combination) reflect the observed SFG intensities in the laboratory frame They are related to ð2Þ ð2Þ vyyz and vzzz as follows: 2ị 2ị vssp ẳ Lyy xịLyy x1 ịLzz x2 Þ sin b2 vyyz ð1Þ ð2Þ ÀLxx ðxÞLxx ðx1 ÞLzz ðx2 Þ cos b cos b1 sin b2 vxxz ð2Þ ÀL ðxÞL ðx ÞL ðx Þ cos b sin b cos b v xx zz xx 2 xzx 2ị vppp ẳ 2ị þLzz ðxÞLxx ðx1 ÞLxx ðx2 Þ sin b cos b1 cos b2 vzxx ỵL xịL x ịL x ị sin b sin b sin b vð2Þ zz zz zz 2 zzz ð2Þ where Lii ðxÞ is a Fresnel coefficient corrected for local fields; and b, b1 and b2 are the angles of the signal, visible and IR beams with respect to the surface normal, respectively For a C3v symmetry point group on an isotropic ð2Þ ð2Þ surface, vxzx ẳ vzxx The Fresnel coefficient Lxx xị can be calculated as follows: Lxx ẳ 2n1 cosht ị ; n1 cosht ị ỵ n2 cosai ị 3ị where and ht are the incident and the transmitted angles of the optical beam, respectively Because the near-totalinternal reflection geometry was used in this study, the The SFG signals are deconvoluted using the Lorentzian line shape function described as follows: X Aq 2ị 2ị veff ẳ vnr ỵ 5ị x xq ỵ iCq IR q 2ị where vnr is the nonresonating contribution; Aq is the amplitude of the vibrational mode q; xIR and xq are the input IR and the resonance IR of the vibrational q mode frequencies, respectively; and C denotes the damping coefficient of the SFG peak Results and Discussions Interaction Between PAP248–286 and 3:7 DPPG/ DPPC Lipid Bilayers The interactions between PAP248–286 and DPPG:DPPC (3:7) lipid bilayers were studied at two peptide concentrations of 200 nM and 1.0 lM This 3:7 lipid ratio is commonly used to simulate a mixed anionic/zwitterionic membrane system It is worth noting that both DPPC and DPPG are in the gel phase at 23 °C, which allows for the SFG spectral specificity of the distal and proximal leaflets using isotope labelling as previously demonstrated by Chen et al (2007) In particular, deuterated 3:7 dDPPG/dDPPC was deposited on to the CaF2 prism as the distal leaflet, and protonated 3:7 DPPG/DPPC was then deposited as the proximal leaflet dDPPC and DPPC were used to reduce the density of negative charges in the lipid bilayer thus lessening the electrostatic interaction between the model membrane and PAP248–286 Despite this lessening of electrostatic interaction, PAP248–286 was bound strongly to the lipid bilayer, demonstrated by the strong SFG amide I band at both peptide concentrations of 200 nM and 1.0 lM (Fig 2) To verify that the interaction between PAP248–286 and DPPG/DPPC lipid bilayer was electrostatically driven, a pure DPPC lipid bilayer was used in place of the 3:7 DPPG/DPPC mixture Results showed that no discernible SFG amide I band was observed (Fig S1, ESI) in the absence of the electrostatic lipid–peptide attraction Despite its strong binding to the membrane, PAP248–286 did not exhibit any ability to penetrate the 3:7 DPPG/DPPC bilayer as demonstrated in Fig Upon time-dependent translocation calibration, the SFG signals of the terminal methyl C–H and C–D stretches suggest that the 3:7 DPPG/ 123 K T Nguyen: In Situ Investigation of Peptide–Lipid Interaction Between PAP248–286… SFG Intensity (A U.) 0.30 200 nM ssp 200 nM ppp 0.20 0.10 0.00 1500 1600 1700 1800 wavenumber (cm-1) SFG Intensity (A.U.) 0.30 μM ssp μM ppp 0.20 0.10 0.00 1500 1600 1700 1800 wavenumber (cm-1) Fig SFG amide I band of 3:7 DPPG/DPPC lipid-bound PAP248–286 at peptide concentration of 200 nM (top) and lM (bottom) in ssp and ppp polarization combinations SFG Intensity (A.U.) (a) 0.10 CH3 sym CH3 FR 0.08 CH3 asym 0.06 0.04 0.02 0.00 2800 caused by PAP248–286 when it was bound to the lipid bilayer Given the fact that PAP248–286 is nondisruptive to the 3:7 DPPG/DPPC bilayer, the main helical segment (G261–I277) should adopt a more or less horizontal orientation at the lipid surface and the observed SFG amide I band should be contributed by the more vertically oriented helical segment K251–G260 This vertically oriented helical segment may play an important role in promoting the bridging interactions between membranes due to its positive charges Since horizontally oriented helical segments were calculated to produce weaker SFG amide I band in both ppp and ssp polarization combinations (Nguyen et al 2009; Wang et al 2008), the strong SFG amide I signals (as compared to that of transmembrane peptide magainin II in POPG/POPC lipid bilayer, Fig S2, ESI) suggest a high helical content of lipid-bound PAP248–286, which is contradictory to the previously reported low value of 30 % suggested by Brender et al using circular dichroism However, the authors concluded that this low value of 30 % was probably an underestimate caused by the visible aggregation of the lipid vesicles (Brender et al 2009) We thus believe that SDS-bound PAP248–286 was denatured by the anionic surfactant SDS as commonly reported in the community (Seddon et al 2004; Warschawski et al 2011) It is worth noting that SDS is a charged soluble detergent which interacts with both the polar and nonpolar regimes of the peptides/proteins Beyond its critical micellar concentration of around 7–8 mM, there coexist both SDS micelles and molecular SDS molecules in the bulk medium, which will at some degree affect the folding of the proteins/peptides in the solution before PAP248-286 aŌer μM PAP248-286 2850 2900 2950 3000 Interaction Between PAP248–286 and 3:7 POPG/ POPC Lipid Bilayers wavenumber (cm-1) SFG Intensity (A.U.) (b) 0.05 0.04 0.03 0.02 before PAP248-286 CD3 asym aŌer μM PAP248-286 CD3 sym CD2 asym CD3 FR 0.01 0.00 2000 2100 2200 2300 wavenumber (cm-1) Fig ssp SFG signals of the proximal (a) and distal (b) lipid leaflets before and after peptide interaction DPPC remained almost unchanged upon the binding of PAP248–286 (Fig 3) The minor spectral differences observed in Fig were most likely due to the minor stress 123 Since both DPPC and DPPG are in gel phase under the current experimental conditions (at room temperature), another set of experiments in which PAP248–286 interacts with lipid bilayers in fluid phase is desirable The 3:7 POPG/POPC bilayer system was chosen for the current study because its electrostatic attraction to PAP248–286 should be similar to that of 3:7 DPPG/DPPC presented in the previous section (Haro et al 2003) With this system, it is impossible to spectrally distinguish the distal and proximal leaflets by the method of isotope labelling of the fluidphase lipid bilayers The flip-flopping rate of fluid-phase lipid bilayers is so rapid [t1/2 can be as low as 1.3 at room temperature (Liu and Conboy 2005)], which almost instantly causes the lipid bilayer to become entirely symmetric, leading to no spectral features being observable by SFG However, being a symmetry-sensitive technique, SFG can probe the symmetry deviation of interfaces SFG K T Nguyen: In Situ Investigation of Peptide–Lipid Interaction Between PAP248–286… (a) 0.30 SFG Intensity (A.U.) SFG Intensity (A.U.) 0.40 0.20 before PAP248-286 aŌer μM PAP248-286 0.10 0.00 2800 2850 2900 2950 3000 0.10 200 nM ppp 200 nM ssp 0.20 (b) 0.30 SFG Intensity (A.U.) SFG Intensity (A.U.) data fit 1605 cm-1 1630 cm-1 1650 cm-1 1685 cm-1 1600 1700 1800 wavenumber (cm-1) Fig 3:7 POPG/POPC lipid bilayer before and after interacting with PAP248–286 0.20 data fit 1605 cm-1 1630 cm-1 1650 cm-1 1685 cm-1 0.10 0.00 1500 0.10 1600 1700 1800 wavenumber (cm-1) 0.00 1500 1600 wavenumber 1700 1800 (cm-1) 0.30 SFG Intensity (A.U.) 0.20 0.00 1500 wavenumber (cm-1) 0.30 0.30 Fig ppp SFG amide I band fitting of lipid-bound PAP248–286 at peptide concentration of lM: a with 3:7 DPPG/DPPC and b with 3:7 POPG/POPC The insets are the zoom-ins to show the smaller contribution peaks μM ssp μM ppp 0.20 0.10 0.00 1500 1600 1700 1800 wavenumber (cm-1) Fig SFG amide I band of 3:7 DPPG/DPPC lipid-bound PAP248–286 at peptide concentration of 200 nM (top) and lM (bottom) in ssp and ppp polarization combinations data reveal that the fluid-phase 3:7 POPG/POPC lipid bilayer exhibits a noticeable decrease in the degree of symmetry upon interacting with PAP248–286, demonstrated by the vibrational modes of the terminal methyl groups starting to appear with the presence of the peptide (Fig 4) This observation can be simply explained by the stresses stemming from the lipid headgroup–PAP248–286 binding It is noted in Fig that the baseline of the SFG signal in the 2800–3000 cm-1 range of the 3:7 POPG/POPC bilayer became weaker after the peptide interaction, which is due in part to the peptide binding that reduces the number of interfacial water molecules On the other hand, it is also possible that the charge neutralization by the peptide binding dictates the interfacial water orientation and leads to the reduction in the SFG signal baseline (Fig S3, ESI) (Ding et al 2013) Further information about the binding behaviour of PAP248–286 to POPG/POPC can be derived by analysing the SFG amide I band of the interfacial peptide molecules Interestingly, both the ssp and ppp SFG amide I signal intensities obtained were similar as occurred when 3:7 DPPG/DPPC was used at peptide concentrations of 200 nM and 1.0 lM PAP248–286 (Fig 5), indicative of a similar nondisruptive binding mode of PAP248–286 stemming from its low hydrophobicity and high positive net charge (?10) Despite the above similarity in the PAP248–286 SFG amide I signal intensities, there were some minor spectral shifts observed that indicate secondary structure content shifts of PAP248–286 upon interacting with the lipids Highresolution technique NMR suggested that PAP248–286 consists of mainly helical (a- or 310-) and random coil structures (PDB codes 2L77 and 2L3H, respectively Figure 1) (Nanga et al 2009) The structure of PAP248–286 in TFE suggested by Ayyalusamy et al (PDB code 2L77) contains significantly more helical content than when the peptide binds to SDS micelles (2L3H) In the current SFG measurements, since SFG is insensitive to unordered structures, random coil components would not make a substantial contribution to the strong amide I band shown 123 K T Nguyen: In Situ Investigation of Peptide–Lipid Interaction Between PAP248–286… Table ppp SFG amide I band fitted amplitude/damping coefficient ratio (A/C) of PAP248–286 peptide interacting with 3:7 DPPG/DPPC and 3:7 POPG/POPC Secondary structure Peak centre (cm-1) 3:7 DPPG/DPPC A/C 3:7 POPG/POPC A/C a-Helix 1650 0.49 0.48 Possibly antiparallel b-sheet (B2 mode) 1630 0.02 0.05 Possibly antiparallel b-sheet (B1 mode) 1685 0.03 0.07 Side chain adsorption of glutamine 1605 0.02 0.02 in Figs and 5, as has previously been computationally (by NLOPredict) and experimentally (by SFG) demonstrated (Ding et al 2013; Nguyen et al 2010b; Wang et al 2008) Besides, random coil structure should give rise to rather broad amide I bands (if observable) due to unpredicted coupling among the backbone C=O units (Fu et al 2011) In this study, the strong SFG amide I bands of PAP248–286 possess well-defined spectral shape that can be fit nicely using three or four component peaks as shown in Fig and Table For these two main reasons, it is safe to assume that the SFG amide I contribution of the random structures of PAP248–286 was negligible in the illustrated spectra Although the amide I band of 3:7 POPG/POPC-bound PAP248–286 was well fit using three peaks featuring the ahelical and b-sheet structures, we could not detect the chiral SFG signal in either psp or spp polarization combination (Fig S1, ESI) Furthermore, our attempt to use the interference method (Belkin et al 2000; Nguyen et al 2010a; Wang et al 2005) to enhance the chiral signals by their interference with the achiral components failed to detect any chiral signal from the lipid-bound PAP248–286 Our inability to probe any chiral signal can be explained either by the non-existence of the b-sheet structure or by the b-sheet content being insufficient to produce any detectable SFG chiral signal We believe the latter explanation is more sensible because a small b-sheet conformation does exist in the structure of PAP248–286 (residues L283–Y286) and the two minor peaks used to fit the achiral spectrum (Fig 6b; Table 1) closely match the assignments of the B2 and B1 vibrational modes previously reported (Baio et al 2013; Nguyen et al 2010a) Whilst the existence of a small and transient/dynamic 310-helical segment at the C terminus has been proposed in an NMR study on SDS-bound PAP248–286 (Brender et al 2009), there was no 310-helical SFG signal detected in our study The small peak at 1630 cm-1 could not have been contributed by the 310-helical structure because this structure would also give rise to a peak at 1670 cm-1 when coupled with the a-helical structures (Ye et al 2012, 2010) It is noted that the 310-helix was not observed in the structure of PAP248–286, either (PDB code 2L77) 123 Since PAP248–286 does not penetrate into the hydrophobic core of lipid bilayers, its molecular orientation at the lipid interface can be reasonably assumed to be independent of the phase of the lipid bilayers The higher SFG signal amplitudes of the 1630 and 1685 cm-1 peaks observed when PAP248–286 binds to fluid-phase lipids can be attributed to the slight peptide conformational/orientational alteration induced by the more intimate binding PAP248–286 causes to the lipid This peptide conformational/orientational alteration demonstrates that the conformational preferences of the monomeric PAP248–286 are rather sensitive to the peptide/lipid interaction, which may provide important insights into the aggregation pathways and the formation of the eventual amyloid fibre Conclusion This study used sum frequency generation vibrational spectroscopy (SFG) to show that the molecular structure of the lipid-bound PAP248–286 may vary with the phase of lipid bilayer system Furthermore, PAP248–286 was observed to interact more intimately with the 3:7 POPG/ POPC lipid bilayer than with its gel-phase counterpart, causing the lipid bilayer to become asymmetric The discrepancies between our results and NMR measurements on SDS-bound PAP248–286 might be due to the choice of 3:7 POPG/POPC lipid bilayer versus SDS micelles used to mimic the cell membranes, which brings into question whether SDS micelles are a good system for such studies The current study also suggests a potentially higher helical content of lipid-bound PAP248–286 than the 30 % calculated using circular dichroism In addition, this study demonstrates the importance of the choice of lipid–bilayer system when conducting studies on the aggregation pathways and amyloid fibre formation of the SEVI precursor peptide PAP248–286 Acknowledgments This research is funded by the Vietnam National Foundation for Science and Technology Development (NAFOSTED) under Grant Number 106.16-2012.67 The author sincerely thanks Dr Gay Marsden for her generous assistance in the manuscript preparation K T Nguyen: In Situ Investigation of Peptide–Lipid Interaction Between PAP248–286… Compliance with Ethical Standards Conflict of interest interests The authors declare no competing financial References Arnold 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