Article pubs.acs.org/JPCC
Phosphate Ions Promoting Association between Peptide and Modeling Cell Membrane Revealed by Sum Frequency Generation Vibrational Spectroscopy Feng Wei, Shuji Ye,* Hongchun Li, and Yi Luo Hefei National Laboratory for Physical Sciences at the Microscale and Department of Chemical Physics, University of Science and Technology of China, Hefei, Anhui, P.R. China 230026 S Supporting Information *
ABSTRACT: Phosphate ion is one of the most important anions present in the intracellular and extracellular fluid. It can form strongly hydrogen-bonded and salt-bridged complexes with arginine and lysine to activate the voltage gated channel protein. A molecular-level insight into how the phosphate anions mediate the interaction between peptides and cell membrane is critical to understand membrane-bound peptide actions. In this study, sum frequency generation vibrational spectroscopy (SFG-VS) has been applied to characterize interactions between mastoparan (MP, a G-protein-activating peptide) and different charged lipid bilayers in situ. It is found that phosphate ions can greatly promote the association of MP with lipid bilayers and accelerate the conformation transition of membrane-bound MP from aggregation into α-helical structure. In phosphate buffer solution, MP can insert not only into negatively and neutrally charged lipid bilayers but also into positively charged lipid bilayers. In neutrally and negatively charged lipid bilayers, the tilt angle of α-helical structure becomes smaller with increasing buffer concentration, while MP adopts a multiple orientation distribution in the positively charged lipid bilayer. MP interacts with lipid bilayers in the salt solution environment most likely by formation of toroidal pores inside the bilayer matrix. Results from our studies will provide insight into the MP action mechanism and offer some ideas to deliver exogenous protein into the cytosol.
1. INTRODUCTION Peptide−membrane interactions play a key role in many biological activities and functions including ion transport, signal regulation, immune response, and membrane assembly.1−3 The interactions are quite complex, which are driven by different peptide−membrane forces such as van der Waals, hydrophobic, hydrogen bonding, and electrostatic forces. The ionic strength is one of the most important parameters for the driving forces. On the other hand, the human body contains about 70% water on average. Most of the water in the human body is contained inside the cells. However, this body fluid is not pure water. It is actually a salt solution containing 1% salts in ionic form.4 Therefore, it is important to study the peptide−membrane interactions in salt aqueous environments rather than in pure water environment in order to understand the nature of the driving force behind such interactions. Phosphate is one of the anions present in the intracellular and extracellular fluid. The concentration of phosphate is of the order of several hundreds of micromolar.5,6 There is evidence in the literatures suggesting that phosphate forms strongly hydrogenbonded and salt-bridged complexes with arginine to activate the voltage gated channel proteins.7−12 Recently, Ye et al. applied sum frequency generation vibrational spectroscopy (SFG-VS) to investigate the interactions between alamethicin and neutral © XXXX American Chemical Society
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayer with the presence of phosphate anions and observed a channel gating action of alamethicin in model cell membranes.13 Although phosphate buffer has been widely used in many biological experiments, the influences of phosphate anions on the protein−membrane interaction are still poorly understood at the molecular level.3 In this research, we used mastoparan (MP) as a modeling peptide and applied SFG-VS to investigate how the phosphate anions mediate the interaction between peptides and cell membrane and thus affect the peptide actions. MP is a 14-residue amphipathic peptide extracted from wasp venom, having the sequence of Ile-Asn-Leu-Lys-Ala-Leu-AlaAla-Leu-Ala-Lys-Lys-Ile-Leu-NH2.14 It has been shown to exhibit various biological activities including mast cell degranulation,15 calmodulin binding,16 G-protein activation,17 stimulation of phospholipases,18 and facilitation of the opening of the mitochondrial permeability transition pore.19 Since G-proteins are located on the inner face of the plasma membrane and associated with intracellular membranes,20 MP must penetrate into the cells to activate G-protein. In fact, ion channel formation of MP in Received: January 11, 2013 Revised: April 11, 2013
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that, they were rinsed with deionized (DI) water before soaking in methanol for 10 min. All of the prisms were then rinsed thoroughly with an ample amount of DI water and cleaned inside a Harrick plasma chamber for 10 min immediately before depositing lipid molecules on them. Substrates were tested using SFG, and no signal from contamination was detected. Single lipid bilayers were prepared on CaF2 substrates using Langmuir−Blodgett and Langmuir−Schaefer (LB/LS) methods with a KSV mini trough LB system. The detailed procedure was similar to previous reports.13,54 The bilayers were immersed in pure water inside a 2 mL reservoir throughout the entire experiment, and a small amount of water could be added to the reservoir to compensate for evaporation when needed for long time scale experiments. For the experiments of the interaction
planar lipid membranes has been reported at high ionic strength (>0.3 M).21 Therefore, a detailed characterization of interactions between MP and model cell membranes in salt solution will be essential to understand the expression of MP activities. Generally, an α-helix composed of 14 amino acid residues is about 2.1 nm long, which is apparently shorter than the hydrophobic thickness of the phospholipid bilayer (3−5 nm);22 thus, an α-helical MP molecule is not long enough to span a phospholipid bilayer. This point naturally poses a lot of questions on the mechanism of MP actions. For example, how does MP interact with lipid bilayers? How does MP penetrate into the cells and activate G-protein? Extensive research has been performed to gain insight into the MP action in cell membranes using a number of techniques, such as nuclear magnetic resonance (NMR) spectroscopy,23−25 circular dichroism (CD),25−27 infrared spectroscopy (IR),26,28 fluorescence,27 and so on. However, the mechanisms still remain elusive so far. Contradictory models for the mechanisms have been proposed: carpet model,29,30 barrel-stave model,29−32 toroidal model,29,31−34 and detergentlike model.35−37 Accumulation on the bilayer surface or formation of a helix bundle has also been suggested.38,39 SFG-VS is a nonlinear optical laser technique which provides vibrational spectra of surfaces and interfaces.40−44 As a polarized vibrational spectroscopy, SFG-VS permits the identification of interfacial molecular species (or chemical groups), and also provides information about the interfacial structure, such as the orientation of functional groups on a surface or at an interface in situ in real time.40−44 SFG-VS has been applied not only to characterize the structure and orientation of various biomolecules (including peptides and proteins) in modeling membrane or other interfacial environments,13,45−52 but also to examine the action mechanism of peptide in lipid bilayers.53 In the present study, we investigated the influence of lipid charge and the phosphate buffer concentration on the peptide amide I stretch signals. It was found that phosphate ions can greatly promote the association of MP into lipid bilayers, even in positively charged lipid bilayers. Results from our studies will provide insight into MP actions and offer some ideas to deliver exogenous protein into the cytosol.
Figure 1. The amide-I ssp spectra of MP molecules when interacting with lipid bilayer in the absence of phosphate buffer solution: (A) DMPG; (B) DMPC; (C) DMEPC. The ssp spectra of DMEPC bilayer itself were also shown in part D for comparison.
2. EXPERIMENTAL SECTION 2.1. Materials and Sample Preparations. MP with a purity of >98% was purchased from Shanghai Apeptide Co., Ltd. The lipids of 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (chloride salt) (DMEPC), 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), 1,2-dimyristoyl(d54)-sn-glycero-3-phosphocholine (DMPC-D54), and 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-racglycerol) (sodium salt) (DMPG) were purchased from Avanti Polar Lipids (Alabaster, AL). The salts of dibasic and monobasic potassium phosphate (K2HPO4 and KH2PO4) were ordered from Aldrich with a purity of >99.8%. Right-angle CaF2 prisms were purchased from Chengdu Ya Si Optoelectronics Co., Ltd. (Cheng Du, China). All of the chemicals were used as received. The stock salt solution with a pH of ∼7.0 was prepared by dissolving K2HPO4 and KH2PO4 into ultrapure water from a Milli-Q reference system (Millipore, Bedford, MA). MP was dissolved in methanol (purchased from Sinopharm Chemical Reagent Co., Ltd.) at a concentration of 2 mg/mL and stored at 3 °C. The phospholipid was dissolved in chloroform (purchased from Sinopharm Chemical Reagent Co., Ltd.) and kept at −20 °C. CaF2 prisms were thoroughly cleaned using a procedure with several steps: They were first soaked in toluene for at least 24 h and then sonicated in soap detergent solution for 0.5 h. After
Figure 2. The time dependence of ssp spectra of MP molecules (25 mg/mL) when interacting with DMPC lipid bilayer in the absence of phosphate buffer solution: (A) 0 h; (B) 0.5 h; (C) 1 h; (D) 3 h; (E) 12.5 h; (F) 13 h. B
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3. RESULTS AND DISCUSSION 3.1. The Interaction between MP and the Lipid Bilayer in the Absence of Salts. The molecular structures of MP in neutrally and negatively charged bicelles/vesicles have been studied in the absence and presence of salts by CD and NMR measurements.23−27,57 Here, we investigated the interaction of MP with three kinds of lipids (DMPG, DMPC, and DMPEC) bilayers, which have negatively, neutrally, and positively charged head groups, respectively. Figure 1 shows the ssp spectra of MP in the absence of salts. The spectra were collected in 3 h after 30 μL of MP solution (2 mg/mL in methanol) was injected into the water subphase (∼2.0 mL) of the lipid bilayer. All of the spectra are dominated by a single resonance peak centered at 1665−1675 cm−1 (Table S1 in the Supporting Information), indicating MP adopts the turn structure on the bilayer surface. Similarly, a MP analogue (mastoparan X) has been observed to aggregate in the vesicles made of POPC/POPG (1:1) lipid in pure water by CD measurements.57 To evaluate the MP structure change with time, we monitored the time-dependent change of the MP amide I signal when MP molecules interact with the DMPC bilayer. As shown in Figure 2, the peak center is at 1665−1675 cm−1 in the first several hours and then shifts to 1655 cm−1 after 10 h (Table S2 in the Supporting Information). The 1655 cm−1 peak is indicative of α-helical structure or random structure.58−60 Previous studies investigated by infrared spectra and CD and NMR measurements indicated that MP molecules transit from random coil to α-helix upon binding to a membrane.23−27,57 In addition, on the basis of the detailed data analysis regarding the SFG signals from α-helical and random coil structures,61 we can conclude that the 1655 cm−1 peak arises from α-helical structure, rather than random coil structure. The result suggests that it takes about 10 h for MP to transform from turn structure into α-helical structure in neutrally charged DMPC bilayer. 3.2. The Interaction between MP and the Lipid Bilayer in the Presence of Phosphate Buffer Solution. Similar to the experiments in pure water, we prepared the lipid bilayer in phosphate buffer solution with a concentration of 50 mM and pH of ∼7.0. We investigated the influence of the phosphate ions on the membrane-bound MP structure and the interaction
Figure 3. The time dependence of the ssp signal detected at 1655 cm−1 of MP molecules when interacting with DMPC lipid bilayer in the presence of phosphate buffer solution (50 mM).
between MP and lipid bilayers, ∼30 μL of MP solution (in methanol with a concentration of 2 mg/mL) was injected into the reservoir. A magnetic microstirrer was used to ensure a homogeneous concentration distribution of peptide molecules in the subphase below the bilayers. 2.2. SFG-VS Experiments. Details regarding SFG theories and instruments have been reported previously.40−44 The SFG setup is similar to that described in our earlier publications.55,56 In this research, all SFG experiments were carried out at room temperature (24 °C). The incidence angle at the air/water surface was 53° for the IR beam and 63° for the visible beam. The energies of the visible beam and IR beam both were less than 60 μJ. SFG spectra from interfacial peptides with different polarization combinations of ssp (s-polarized SFG output, s-polarized visible input, and p-polarized infrared input) and ppp were collected using the near total internal reflection geometry. The SFG signals were measured after the interaction between MP and lipid bilayers reached equilibrium. All SFG spectra were averaged over 100 times at each point and normalized by the intensities of the input IR and visible beams. The orientation of α-helical structure was determined using SFG amide I spectra. The SFG data analysis methods are presented in the Supporting Information.
Figure 4. The amide-I SFG spectra of MP molecules when interacting with lipid bilayer in the presence of phosphate buffer solution: (A) DMPG; (B) DMPC; (C) DMEPC. C
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Figure 5. The amide-I ssp spectra of MP molecules when interacting with lipid bilayer in the presence of different phosphate buffer concentrations: (A) DMPG; (B) DMPC; (C) DMEPC.
Molecular orientation information can be obtained by relating SFG susceptibility tensor elements χijk (i, j, k = x, y, z) to the SFG molecular hyperpolarizability tensor elements βlmn (l, m, n = a, b, c) via the Euler transformation.62 The details of orientation determination of α-helical structure using the relationship between the measured ppp and ssp amide I spectral intensity ratios (2) (χ(2) ppp/χppp) have been published previously by treating α-helical structure as having C18/5 symmetry by Chen group.51 In terms of the methodology developed by the Chen group,51 the hyperpolarizability tensor elements for the 14-residue α-helix were deduced and have the relationship of βaca = 0.358βccc and βaac = (2) 0.635βccc. A relation between the measured χ(2) ppp/χssp ratio and θ for α-helical structure is thus calculated (Figure S2, Supporting Information). We put the SFG equations and math related to the orientational analysis of α-helix in the Supporting Information, since those have already been published by the Chen group.51 (2) Here the experimentally measured χ(2) ppp/χssp ratios of the peaks at 1655 cm−1 in the DMPG/DMPG, DMPC/DMPC, and DMEPC/DMEPC bilayers are 1.15, 1.35, and 1.2, respectively. (2) As shown in Figure S2 (Supporting Information), the χ(2) ppp/χssp ratios of MP in DMPG/DMPG (1.15) and DMEPC/DMEPC
dynamics. Figure 3 shows the time-dependent change of the ssp amide I signal of the characteristic α-helical band at 1655 cm−1 after MP interacts with DMPC bilayer in 50 mM phosphate buffer solution. In contrast to the slow interaction process in pure water shown in Figure 2, the interaction of MP with DMPC bilayer in phosphate buffer solution reached equilibrium in several minutes and formed an α-helical structure. It is evident that phosphate ions can greatly promote the association between MP and lipid bilayer. Figure 4 shows the ssp and ppp spectra in the amide I region after MP interacted with lipid (DMPG, DMPC, and DMEPC) bilayers and reached equilibrium. All the ssp spectra show one strong peak at ∼1655 cm−1 and a weak peak at ∼1720 cm−1. The 1720 cm−1 signal is generated by the carbonyl groups of the lipid bilayer. In the ppp spectra, in addition to the peaks at ∼1655 and 1720 cm−1, a broad peak at ∼1580 cm−1 appeared. This broad peak arises from the addition of phosphate ions to the subphase.13 In addition, we collected the spp and psp spectra from MP molecules when interacting with lipid bilayer in the presence of phosphate buffer solution. No detectable signals are observed in spp and psp spectra. The formation of β-sheets can be ruled out. D
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(1.2) are below the possible range, indicating the MP has a multiple orientation distribution in these two kinds of lipid bilayers,46 while the tilt angle of θ versus the surface normal is about 15° in the DMPC/DMPC bilayer when a δ-distribution is assumed. It is worth mentioning that, although we assumed the orientation distribution as a δ-distribution, it is in fact very narrow, which has been suggested by the study on alamethicin in lipid bilayers using SFG-VS and ATR-FTIR measurements.13,52 3.3. The Effect of Phosphate Buffer Concentration. To investigate the effect of phosphate buffer solution, we first prepared the lipid bilayer in pure water and then injected ∼30 μL of MP solution (in methanol with a concentration of 2 mg/mL) into the water subphase (∼2.0 mL) of lipid bilayer. The SFG spectra were collected after 2 h of interactions. After that, a certain amount of phosphate buffer solution (in 0.5 M solution) was added into the subphase to achieve the target salt concentration. It was given at least 2 h to allow the interaction between MP and bilayer to reach equilibrium before polarized SFG spectra were collected. Then, more phosphate buffer solution was added to get another target salt concentration. It takes less than 20 min to reach equilibrium in all targeted concentrations. We measured the pH values of the solution for all targeted phosphate concentrations. The pH values all fall in the range 7.0−7.2. Figure 5 shows the ssp amide I signal of MP in DMPG, DMPC, and DMEPC bilayers in different buffer concentrations. All the SFG spectra in buffer solution show a strong peak at ∼1655 cm−1 and a weak peak at ∼1720 cm−1. To quantitatively analyze the change in the intensity of the ∼1655 cm−1 peaks as the buffer concentration varied, we fitted the spectrum using eq (S2) given in the Supporting Information. The fitting amplitudes of the peak at ∼1655 cm−1 (Figure 6) and the experi-
(2) −1 −1 Figure 7. The fitted ratio of χ(2) ppp (1655 cm )/χssp (1655 cm ) and tilt angle of MP molecules when interacting with DMPG, DMPC, and DMEPC lipid bilayers plotted as a function of phosphate buffer concentration. The fitting curve (dotted line) was just used for guiding the readers’ eyes and did not have any physical meaning.
with lipid bilayer and accelerate the conformation transition from turn into α-helical structure in lipid bilayer. As shown above, two different experimental orders have been performed in the current study: (1) the lipid bilayers were prepared in buffer solution before injecting MP; (2) the lipid bilayers were prepared in pure water, and MP was injected prior to the addition of phosphate buffer solution. However, these two orders (2) −1 produced different χ(2) ppp/χssp ratios of the 1655 cm peak for the interaction between MP and DMPG bilayer, while they gave −1 (2) similar χ(2) peak for the ppp/χssp ratios (∼1.2) of the 1655 cm interaction between MP and DMEPC bilayer. The ratio (∼1.2) is below the possible range in Figure S2 (Supporting Information), resulting in a multiple orientation distribution of MP in DMEPC bilayer. For the interaction between MP and DMPC or DMPG (2) bilayer, the χ(2) ppp/χssp ratios decrease with the buffer concentration increasing in the second experimental order. The deduced tilt angles of MP in DMPC and DMPG bilayers are plotted in the bottom panel of Figure 7. With the buffer concentration increasing, the tilt angle becomes smaller. On the basis of this orientational angle change, the interaction between MP and lipid bilayer can be described by Figure 8. 3.4. Interaction Mechanism of MP in DMPC Bilayer and DMPG Bilayer. As mentioned above, the mechanism by which MP interacts with lipid bilayers is controversial: different models have been proposed such as the carpet model,29,30 barrel-stave model, 29−32 toroidal model, 29,31−34 and detergent-like model.35−37 The SFG spectra response to these models is different. In the carpet or detergent-like model, the peptides bind onto the membrane surface by orienting parallel to the surface of the lipid bilayer, predominantly by electrostatic interactions, until a threshold concentration is reached. In the barrel-stave model, the attached peptides aggregate and insert into the hydrophobic core of the membrane and assemble therein in a barrel-stave manner to form transmembrane pores. In the toroidal model, the attached peptides aggregate and induce the lipid monolayers to bend continuously through the pore so that the water core is lined by both the inserted peptides and the lipid head groups.29,63
(2) −1 −1 Figure 6. The fitted amplitude of χ(2) ppp (1655 cm ) and χssp (1655 cm ) of MP molecules when interacting with lipid bilayers plotted as a function of phosphate buffer concentration. The fitting curve (dotted line) was just used for guiding the readers’ eyes and did not have any physical meaning. (A) DMPG; (B) DMPC; (C) DMEPC lipid bilayers.
(2) mentally measured χ(2) ppp/χssp ratios (top panel in Figure 7) were plotted as a function of buffer concentration. In total, the fitting amplitudes of ssp and ppp both follow an exponential growth function. As indicated by Figure 7, even at very low-concentration salt solution, phosphate ions can promote the association of MP
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Figure 8. A scheme of the interaction mechanism of MP in DMPG, DMPC, and DMEPC lipid bilayers under different phosphate buffer concentrations.
energetically unfavorable. However, in phosphate buffer solution, MP can insert into not only negatively and neutrally charged lipid bilayer but also positively charged DMEPC lipid bilayer. This phenomenon was also observed in the interaction between other positively charged peptides (such as mastoparan-X, melittin, and LKα-14) and DMEPC bilayer (Figure 9). This indicates that the peptide charges interact more favorably with their ionic atmospheres when the peptide is located close to the bilayer than they can do in purely water surroundings.65 It is generally believed that the initial interaction between peptide and membrane is driven by electrostatic forces involving the cationic residues of the peptides and the anionic moieties of the membrane. For this case, the addition of phosphate buffer will generate the charge screening effect, which can weaken the repulsion between the positively charged peptides and positively charged lipids, thus favoring MP molecules inserting into the positively charged lipid bilayer. Correspondingly, the screening effect will not favor the interactions between MP molecules and the negatively charged lipids. However, it is opposite to what was observed in the experiment. Therefore, we believe that interaction between MP and lipid bilayer is not dominated by electrostatic force. Why can the phosphate buffer solution promote the association of peptides into lipid bilayer? Earlier NMR study indicated that the amino acid of lysine and arginine can form a strong hydrogen bonding network with the phosphate headgroup on the outer leaf of lipid bilayer.11,66 The lysine (arginine)− phosphate interaction plays a stronger structural role in the translocation of peptides across the fluid-phase bilayer. In the peptides of MP, MP-X, melittin, and LKα-14, their N-terminal part is largely hydrophobic, whereas the C-terminal end exhibits a highly polar nature due to its lysine (or arginine) residues.65 With the addition of phosphate buffer solution, the HPO42− and H2PO4− ions can interact with lysine (or arginine) residues and neutralize the positive charges of peptides and lipids, and thus modify the hydrogen-bonding interaction and hydration environments of lipid bilayer, resulting in the promotion of the insertion of MP into lipid bilayer, which is confirmed by the interaction between MP and lipid bilayers in chaotropic ion environments, as well as the interaction between MP and the lipids without a phosphate group. The detailed study of the influence of different ions on the interaction between MP and lipid bilayer will be presented in the next paper, which will aid in a better understanding of protein−membrane interaction driving force.
Large signals from carbonyl groups and alkyl chains of the lipid bilayer will be generated in the carpet, detergent-like, and toroidal models because the peptides either lie down on the lipid bilayer surface or induce the lipid monolayers to bend continuously, and then the carbonyl groups and alkyl chains were distorted, which makes the signal of these groups from two leaflets not cancel each other. However, no or weak signals from the lipid carbonyl group and alkyl chains will be observed in the barrel-stave model, since peptides directly disrupt the lipid bilayer. Our results show that MP inserts into DMPC or DMPG bilayers in the presence of the phosphate buffer solution with a tilt angle. The tilt angle becomes smaller with increasing phosphate buffer concentration. In addition to the amide I signal at ∼1655 cm−1, a weak peak at ∼1720 cm−1 from the lipid carbonyl group was observed in Figures 4 and 5. In terms of the definition of these several models, the toroidal model is the most likely one in a salt solution environment, since other models can be ruled out, while the carpet or detergent-like model is the possible one in pure water surrounding. The conclusion is further confirmed by monitoring the structure change of DMPC-D54 bilayer shown in Figures S5 and S6 in the Supporting Information. The hydrocarbon interior of DMPC-D54 is deuterated, while the headgroup is hydrogenated. Thus, we can distinguish the behaviors of lipid hydrocarbon and MP during the interaction between MP and lipid bilayers.64 Before injecting MP molecules, very weak signals at 2075 and 2215 cm−1 were observed (Figure S5A in the Supporting Information). The peaks at 2075 and 2215 cm−1 originate from symmetric stretch and asymmetric stretch of the CD3 group separately.42 Weak C−D signals from isotopically symmetric bilayers indicate that the proximal leaflet and distal leaflet had very similar structures and signals generated from both leaflets effectively canceled each other. After injecting MP molecules, we measured the ssp spectra in the frequency range 2000−2300 cm−1 again (Figure S5B−H in the Supporting Information). The signal at 2070 cm−1 becomes much stronger, which indicated that the alkyl chains were strongly perturbed by MP molecules. This effect is most likely caused by the penetration of MP molecules into the hydrophobic hydrocarbon region of the lipid bilayer outer leaflet. Thus, the signals generated from both leaflets cannot cancel each other. The fitted amplitude of CD3 symmetric stretching as a function of time (Figure S6 in the Supporting Information) follows the similar trend with the amide I signals shown in Figure 3. 3.5. Further Discussion on the Phosphate Ion Effect. MP is a positively charged peptide. Generally, the insertion of positively charged peptide into positively charged lipid bilayer is F
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Figure 9. The amide-I SFG spectra of peptide molecules when interacting with DMEPC lipid bilayer in the presence of different phosphate buffer concentrations: (A) melittin; (B) MP-X; (C) LKα14.
Notes
4. CONCLUSION We have applied sum frequency generation vibrational spectroscopy to characterize interactions between mastoparan and different charged lipid bilayers in situ. It is found that phosphate ions can greatly promote the association of MP with lipid bilayer and accelerate the conformation transition of membrane-bound MP from the turn structure into α-helical structure. In phosphate buffer solution, MP can insert not only into negatively and neutrally charged lipid bilayers but also into positively charged lipid bilayer. In neutrally and negatively charged lipid bilayers, the tilt angle of α-helical structure becomes small with the buffer concentration increasing, while MP adopts a multiple orientation distribution in positively charged lipid bilayer. MP interacts with lipid bilayer in salt solution environment most likely by formation of toroidal pores inside the bilayer matrix.
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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REFERENCES
This work was supported by the National Basic Research Program of China (Grant No. 2010CB923300), the National Natural Science Foundation of China (Grant Nos. 21273217 and 91127042), and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry.
(1) Grasierger, B.; Minton, A. P.; Delisi, C.; Metzger, H. Interaction between Proteins Localized in Membranes. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 6258−6262. (2) Engel, M. F. M.; Khemtémourian, L.; Kleijer, C. C.; Meeldijk, H. J. D.; Jacobs, J.; Verkleij, A. J.; Kruijff, B.; Killian, J. A.; Höppener, J. W. M. Membrane Damage by Human Islet Amyloid Polypeptide through Fibril Growth at the Membrane. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 6033−6038. (3) Killian, J. A.; Nyholm, T. K. M. Peptides in Lipid Bilayers: the Power of Simple Models. Curr. Opin. Struct. Biol. 2006, 16, 473−479. (4) Sizer, F. S.; Whitney, E. N. Nutrition: Concepts and Controversies; Wadsworth Cengage Learning: Independence, KY, 2010. (5) Pradhan, P.; Ghose, R.; Green, M. E. Voltage Gating and Anions, Especially Phosphate: A Model System. Biochim. Biophys. Acta 2005, 1717, 97−103.
ASSOCIATED CONTENT
S Supporting Information *
Details about SFG data analysis procedures, some ppp SFG spectra, and the fitting parameters of Figures 1 and 2. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Fax: 086-551-63603462. Phone: 086-551-63603462. G
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