MBP was extracted from bovine brain and purchased from Merck (Darmstadt, Germany). MBP has been diluted against pure water while at the same time used as the working solution in the concentration of 5 × 10⁻⁹ M. 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoserinesodium salt (DPPS) from bovine heart was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA) and used without further purification. Spreading solutions of DPPC and DPPS were prepared in chloroform/methanol 3:1 (v/v) mixture at a concentration of 0.1 mg/mL. For the mixed lipid solution, DPPC and DPPS were used in a volume ratio of 7:3, which is the ratio of these lipids typically found in the plasma membrane. The ultrapure water used as subphase was distilled and purified with a Millipore purification system (electrical resistivity ≥ 18.2 MΩ·cm).

Surface pressure–area (π–A) isotherms were measured on a KSV Minitrough system (Helsinki, Finland). It was operated on a Wilhelmy platinum plate with a dynamic surface pressure range of 0–150 mN/m and a resolution of 0.01 mN/m.

Monolayers have been formed by spreading an aliquot of lipid solution on 10 mM Tris (hydroxyethyl) amino-methane titrated to pH 7.2 with HCl. Certain volumes of the lipid solutions were spread on the subphase containing the moderate MBP using a Hamilton micro syringe. 15 minutes were allowed for evaporation of the chloroform to occur. The isotherm for DPPC/DPPS was then recorded from online measurements and plotted by compressing the barriers at a forward rate of 10 mm/min. The temperature of the subphase was maintained within 20±1° with the aid of a water circulator bath. The trough and barriers were thoroughly cleaned with absolute ethyl alcohol and ultrapure water between each separate isotherm.

Compression of the DPPC monolayer occurred until the target surface pressure of 10 mN/m was reached. Monolayers have been transferred to the surface of mica substrate (transfer ratio is 1) with a method of vertical pulling transferring. In order to keep the area of the monolayers, pressure which changes with time has been recorded so as to obtain π–T isotherms when the lipid monolayer is compressed to a surface pressure. The whole experiment was repeated three times.

AFM images were acquired in air at room temperature using a SPM-9500-J3 AFM (Shimadzu Corporation, Japan), which provided both a topographical image and a phase contrast one. The sample imaging has been carried out in the contact mode through a Micro-V-shaped Cantilever probe (Olympus Corporation, Japan) with cantilever spring constant about 0.06 N/m. A 55 μm×55 μm tube-type piezoelectric scanner and a Si₃N₄ probe were used and images in height mode were collected simultaneously with 512×512 points with scan rate of 1.0 Hz per line.

The LB techniques become perceived as desirable model systems for investigating the important role of the interaction between lipids and proteins in the structure and function of biological cell membrane. The typical pressure–area isotherm can be written as a two-dimensional van der Waals equation of state analogous to the three-dimensional real gas^[33,34]

To get insight into the physical state of the investigated monolayer, we first discussed the π–A isotherms. It is possible to determine the dilational elasticity, under quasi-static compression of surface area. The values of the foregoing parameters were calculated from the isotherm data points, at a given monolayer composition^[36]

Figures 2(a), 2(b), and 2(c) show that the interactions of MBP with different lipid monolayers (DPPC, DPPC/DPPS, and DPPS) were characterized by measuring the π–A curves before and after the protein adsorption at the air–subphase interface under physiological conditions (Tris-HCl buffer at pH 7.2). Figure 2(a) reports the π–A isotherms obtained for DPPC monolayer spread on buffer without or with MBP (concentration: 5 nM). The monolayer of DPPC shows the presence of liquid expanded–liquid condensed (LE–LC) phase transition plateau at about 3–6 mN/m, and collapses at pressure higher than 65 mN/m, which is similar to previous results obtained by others.^[38,39] For mixed DPPC-MBP monolayers, the curve was shifted to a larger area per molecule when the concentration of the MBP is 5 nM. Compared with the phase transition of pure DPPC monolayer, an increase of mixed MBP/DPPC monolayer phase transition occurred for surface pressures higher than 3 mN/m. MBP has a high positive charge, making the π increase at the LC–LE phase coexistence for DPPC (see insert of Fig. 2(a)). This means that the MBP molecules penetrate the monolayer and interact with the phospholipid at about 10 mN/m.

Fig. 2.

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	Fig. 2. π–A isotherms of lipid monolayers in the absence and in the presence of MBP in the subphase (5 nM): (a) DPPC, (b) DPPS, and (c) DPPC/DPPS mixed monolayers. Inset: compressibility modulus as a function of area per molecule for the same compositions.

The isotherms of DPPS monolayer measured in the presence of MBP are shown in Fig. 2(b), where the curve is mainly shifted toward higher molecular area than the isotherm of pure DPPS. At lift-off, the curve with the MBP mixtures shows higher mean molecular area (MMA) of 149.5 ±1.0 Å², followed by the pure DPPS at 61.5±1.5 Å². The monolayer of DPPS shows the presence of liquid expanded–liquid condensed (LE–LC) phase transition plateau at about 10–20 mN/m. It can be seen in the inset of Fig. 2(b) that when the surface pressure is lower than 38 mN/m, the compression modulus of the mixed film is slightly lower than pure DPPS monolayer. In contrast, when the surface pressure is greater than 38 mN/m and less than 70 mN/m, DPPS pure compressive modulus is slightly higher in the mixed monolayer. This means pure DPPS cannot easily be compressed when the surface pressure is lower than 38 mN/m. When the surface pressure is greater than 38 mN/m, the protein is absorbed onto the membrane lipid monolayer, the monolayer is so difficult to compress. This implies that MBP will remain combined with the DPPS monolayer at high surface pressure.

A qualitatively similar behavior was observed with the mixed monolayer of DPPC/DPPS (7:3, w/w) containing MBP. DPPC/DPPS mixed membrane has not undergone liquid-expanded to liquid-condensed transition in the plateau-like region between 60 and 38 Å². This result is consistent with the insert of Fig. 2(c). The area increase is larger in the presence of MBP than for DPPC/DPPS, and the isotherm of the mixed monolayer is parallel to that of DPPC/DPPS mixed.

From experimental results shown in Figs. 2(a), 2(b), and 2(c) we find that the isotherms shift to a larger area, due to the interaction between MBP and lipids. From versus π plot of the DPPC and DPPC/DPPS monolayer containing the moderate MBP, we observe that the mixed monolayer membrane elastic modulus is higher than pure lipid compression modulus. The higher compression modulus makes it more difficult for the monolayer to be compressed. This shows that the pure lipids membrane has good liquidity, i.e., easily compressed, whereas the monolayer cannot easily be compressed when the protein is absorbed onto the lipid monolayer. This behavior indicates that the MBP molecules have been bound to the lipids.

In order to study the relation of MBP molecules with DPPC, DPPC/DPPS, and DPPS monolayers spreading over the air–water interface, we measured the surface pressure-time (π–T) isotherms and determined the insertion ability of MBP into lipid monolayer in the presence of lipids (DPPC, DPPC/DPPS, DPPS) at three initial surface pressures (π_initial) equal to 10, 20, and 30 mN/m. Insertion can be measured by the increase in surface pressure (Δπ = π_final − π_initial). Previous research^[40] has shown that the monolayer is not affected by surface pressure without protein insertion into the lipid monolayer. Figure 3 shows an increase of surface pressure, as the DPPC, DPPC/DPPS, and DPPS are spread over the interface with an initial surface pressure of 20 mN/m. We find the absorbed behaviors on the three types of lipid monolayers in the order of DPPC>DPPC/DPPS>DPPS. This implies that the strength of protein–lipid interactions is not the only determinant of the lipid head groups, and the value of the surface pressure can also be used to analyze the extent of protein/lipid interactions.^[41] Table 1 shows that the larger an initial surface pressure is, the smaller the increase in surface pressure is, which means that MBP was mainly in the LE phase embedded in the lipid monolayers. For the lipid molecules arranged more loosely, with lower surface pressure, the MBP is easily inserted into monolayers.

Fig. 3.

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	Fig. 3. Insertion of the MBP into lipid monolayer π–T isotherms. Variation of surface pressure with time after spread DPPC, DPPC/DPPS, and DPPS over the same subphase concentration, which was spread at the initial surface pressure of 20 mN/m.

Table 1.

Table 1.

Table 1. The increases in surface pressure (Δπ) of the DPPC, DPPC/DPPS, and DPPS monolayers at different initial surface pressures (πinitial) of the monolayers at the same subphase concentration of 5 nM. . πi/mN·m−1 DPPC (Δπ) DPPC/DPPS = 7/3 (Δπ) DPPS (Δπ) 10.00 3.84 1.30 0.30 20.00 1.99 0.90 0.18 30.00 1.03 0.52 0.10

Table 1. The increases in surface pressure (Δπ) of the DPPC, DPPC/DPPS, and DPPS monolayers at different initial surface pressures (πinitial) of the monolayers at the same subphase concentration of 5 nM. .

The lipid monolayer is an interesting and useful model membrane system for investigating the three parameters (Δπ, synergy, and maximum insertion pressure (MIP)) responsible for a particular protein with different lipid monolayers.^[42,43] To characterize MBP adsorption and lipid specificity without the need of radio labels or other tags, figure 4 shows the plot of Δπ as a function of π_initial. The Δπ and MIP can be determined by extrapolating the regression of the plot to the y axis and x axis, respectively. The term “synergy” has been calculated because the slope of the linear regression+1 corresponds to the synergy of the monolayer at which the interaction of MBP with the lipids is energetically favorable. In addition, the synergy factor provides additional information on the insert of protein for the lipid monolayers. The value of negative synergy suggests that the protein disfavors combination to the lipid monolayers. In contrast, the positive synergy means the strong interaction between protein and lipids. On the basis of the relationship between Δπ and π_initial, the linear regression for MBP in the presence of DPPC, DPPC/DPPS (7:3, v/v), and DPPS monolayers, results in

Fig. 4.

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	Fig. 4. The binding parameters of MBP onto the DPPC, DPPC/DPPS, and DPPS monolayers determined by plot of Δπ as a function of πinitial. The subphase concentration of 5 nM, pH = 7.2.

The largest values of MIP 40.00±1.80 mN/m have been obtained for the MBP combined to the DPPS monolayer which also suggested the greatest ability to remove the DPPS from the cell membrane, followed by DPPC/DPPS and DPPC monolayers. Values of synergy factor of 0.99±0.01, 0.94±0.02, and 0.86±0.01 have been respectively obtained for DPPS, DPPC/DPPS, and DPPS monolayer which correlate with a favorable binding of MBP to these phospholipid monolayers. Therefore, the values of Δπ, synergy, and MIP depend on the lipid head groups and initial surface pressures of phospholipid monolayers. The fact that the highly positively charged MBP could aggregate is adsorbed onto the net negatively charged DPPS and the zwitterionic phospholipids via electrostatic and hydrophobic interactions.

This partly has demonstrated that the MBP penetrates very slightly into DPPC monolayer. (i) When the MBP concentration in the subphase is 5 nM, the average molecular area increase of the DPPC monolayer reaches ∼ 50 Å², followed by DPPC/DPPS (∼ 70 Å²) mixed and DPPS (∼ 80 Å²) monolayers. (ii) MBP insert, into DPPS and DPPC/DPPS monolayers suggesting that the presence of MBP induces phase separation and perturbation of lipid configuration. Instead, MBP had no significant effect on the surface morphology of DPPC monolayer. (iii) From three parameters of linear equation we can see that MBP interacts preferentially with the net negatively charged DPPS followed by DPPC/DPPS, and finally DPPC monolayers.

To further characterize the effect of the mixture of MBP on the DPPS, DPPC/DPPS (7:3, V/V), and DPPC monolayers, AFM was employed to view selected surface morphologies of the mixture of the different lipid monolayers and MBP deposited on mica substrates at low (10 mN/m), intermediate (20 mN/m), and high (30 mN/m) surface pressure. Figure 5 shows images of the effect produced by the MBP at various initial surface pressure. Therefore, there are two aspects to understanding the morphologies observed. Firstly, how the molecules orient themselves at the air–subphase interface when the monolayers are deposited at various surface pressures. In the presence of MBP, with the increase of surface pressure, the mixture structure of monolayers yielded a densely packed structure and holes are clearly visible, regardless of the lipid conformational change (Figs. 5(c), 5(f), and 5(i)). It implied MBP is squeezed out of the monolayer at high surface but remains associated to the interface, which is consistent with the π–T isotherm shown in Fig. 3. Furthermore, the incorporation of MBP into the lipid monolayers had exerted a significant effect on the domain structure (Figs. 5(b), 5(e), and 5(h)) at a given surface pressure (e.g., 20 mN/m in this case). We found that MBP induces the phase segregation, the mixture of DPPS/MBP forms small watercress sheet domains and the MBP embedded in it, with the height of around 12 nm. The mixture of DPPC/DPPS monolayers in the presence of MBP forms two-phase morphology, one is homogeneous sheet structure from DPPC/MBP monolayer, another is watercress linear region from DPPS/MBP. This behavior is consistent with previous studies on the π–A curves of the interaction between DPPS, DPPC/DPPS, and MBP at 10–20 mN/m. For DPPC/MBP monolayer the formation of relatively loose micro petals is indicated, along with the existence of granular-like domains (Fig. 5(g)). The attraction and binding of MBP to monolayers of different lipid compositions is modulated by hydrophobic interactions and electrostatic interactions with the amphiphilic and acidic lipid headgroups.

Fig. 5.

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	Fig. 5. AFM image (10×10) of DPPS (a)–(c), DPPC/DPPS (7:3, V/V) (d)–(f), and DPPC (g)–(i) in the presence of MBP (5 nM) at low, medium, and high surface pressure: 10 mN/m ((a), (d), (g)), 20 mN/m ((b), (e), (h)), 30 mN/m ((c), (f), (i)). Scale bar: 5 μm.