† Corresponding author. E-mail:
‡ Corresponding author. E-mail:
Project supported by the National Natural Science Foundation of China (Grant Nos. 21402114 and 11544009), the Natural Science Basic Research Plan in Shaanxi Province of China (Grant No. 2016JM2010), the Fundamental Research Funds for the Central Universities of China (Grant No. GK201604004), and the National University Science and Technology Innovation Project of China (Grant Nos. 201610718014 and cx16018).
Myelin basic protein (MBP) is an essential structure involved in the generation of central nervous system (CNS) myelin. Myelin shape has been described as liquid crystal structure of biological membrane. The interactions of MBP with monolayers of different lipid compositions are responsible for the multi-lamellar structure and stability of myelin. In this paper, we have designed MBP-incorporated model lipid monolayers and studied the phase behavior of MBP adsorbed on the plasma membrane at the air/water interface by thermodynamic method and atomic force microscopy (AFM). By analyzing the pressure–area (π–A) and pressure–time (π–T) isotherms, univariate linear regression equation was obtained. In addition, the elastic modulus, surface pressure increase, maximal insertion pressure, and synergy factor of monolayers were detected. These parameters can be used to modulate the monolayers binding of protein, and the results show that MBP has the strongest affinity for 1,2-dipalmitoyl-sn-glycero-3- phosphoserine (DPPS) monolayer, followed by DPPC/DPPS mixed and 1,2-dipalmitoyl-sn-glycero-3-phospho-choline (DPPC) monolayers via electrostatic and hydrophobic interactions. AFM images of DPPS and DPPC/DPPS mixed monolayers in the presence of MBP (5 nM) show a phase separation texture at the surface pressure of 20 mN/m and the incorporation of MBP put into the DPPC monolayers has exerted a significant effect on the domain structure. MBP is not an integral membrane protein but, due to its positive charge, interacts with the lipid head groups and stabilizes the membranes. The interaction between MBP and phospholipid membrane to determine the nervous system of the disease has a good biophysical significance and medical value.
Biological membrane is a supramolecular structure constituted by phospholipids and proteins, which are essential components for cellular activities.[1–3] Biological membranes have molecule alignment similar to crystal structure, and have the fluidity of liquid.[4–7] Therefore, the bio-membrane is a typical structure of the liquid crystalline state. Biological membrane of the lipid content and water system that lyotropic liquid crystal containing two components.[8,9] Here, we mainly focus on the lyotropic liquid crystal, which is regarded as the simple model of biological membrane: the system of an amphiphilic molecule and water. On the one hand, the monolayer of an amphiphilic may be considered as half of a membrane and is the simplest model system that can be discussed.[10,11] On the other hand, the monolayer of a liquid crystal on air–water interface can reveal the richness of phases and an amazing diversity of structures, which plays an important role in living cells.[12] Various techniques of membrane mimicking have been developed. Among these the measurement of pressure–area curves of a Langmuir monolayer is most often used.[13,14] The relationship between bio-membrane with liquid crystal has been noticed.[9] When people study the structure of living cells, myelin shape has been described as liquid crystal structure.
Myelin is the insulating, multi-lamellar membrane discontinuously wrapped around the nerve axon. Myelin integrity is the foundation of the fast salutatory conduction of the signal along the axon in the central nervous system (CNS). One of the major CNS compact myelin proteins is MBP, which accounts for about 30%.[15–17] The main physiological role of MBP is to maintain the structural integrity of the myelin sheath via adhesion to cytoplasmic leaflets of the oligodendrocyte membrane.[18] MBP has vital significance in many neurological diseases,[19–21] such as multiple sclerosis (MS) and experimental cerebral spinal cord inflammation (EAE). A great deal of research[22,23] has demonstrated that the major isoform of MBP in mature myelin has a molecular weight of 18.5 kDa and a net positive charge of 19. MBP has a high net positive charge which is likely to be crucial to its interaction with the phospholipids in model membranes. Numerous studies have now shown that MBP causes adhesion of the cytosolic surfaces and maintain the structural integrity of myelin by binding to phospholipids contain electrostatic and hydrophobic.[24] That is, minor changes in lipid composition in myelin membrane will alter myelin membrane domain structure, size, and the adsorption characteristics of the intermembrane. Therefore, the study of the interaction mechanism of MBP with different lipid compositions is important in understanding the myelination process.
To better study the detailed interaction mechanism, we use a monolayer of myelin lipids at the air–water interface to mimic the actual myelin membrane. Recently, we characterized the adsorption of MBP into model lipid monolayers, composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS), as representative of neutral and charged lipids. The structures are shown in Fig.
Adsorption of MBP onto phospholipid monolayers at the air–subphase interface has been extensively studied in the past by Langmuir–Blodgett (LB) techniques, atomic force microscopy (AFM), scanning electron microscope (SEM), surface forces apparatus (SFA), immune-electron microscopy, and x-ray diffraction.[15,25–28] Some studies[29] demonstrated the importance of hydrophobic interaction between MBP and lipids. Other studies[30,31] have shown a temporal evolution of the structure of subphase penetrating the lipid phase together with the protein. In these studies, the possible origin and regulation of the domain segregation induced by MBP was previously observed in monolayers. In particular, the interface parameters (adsorption time, surface pressure, and type of lipid) behavior of MBP with lipids by LB techniques would be studied. The method enables us to visually provide information to better understand the interfacial behavior of MBP with lipids.
In this paper, we have concentrated on the change of the lateral organization induced by the MBP and lipids (DPPC, DPPC/DPPS, DPPS) when spread as a monolayer at the air–subphase interface. Here, we have used Langmuir–Blodgett (LB) technology and atomic force microscopy (AFM)[32] to investigate the membrane interaction of MBP and lipids. The lipid monolayers were formed in a Langmuir trough at various initial surface pressures (πinitial). Although there have been many reports on the interaction of MBP and lipids, as far as we are aware, this is the first study on the interaction of MBP and DPPC/DPPS (the volume ratio of 7:3) at different initial surface pressures. The measurement of the maximum insertion pressure (MIP) can provide additional useful information for evaluating the binding parameters of MBP such as the synergy. In this article, the procedure to determine the Δπ, MIP, and synergy will be described and the experimental values of Δπ, MIP, and synergy obtained with MBP will be presented and discussed.
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,
Figures
The isotherms of DPPS monolayer measured in the presence of MBP are shown in Fig.
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.
From experimental results shown in Figs.
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 (πinitial) equal to 10, 20, and 30 mN/m. Insertion can be measured by the increase in surface pressure (Δπ = πfinal − πinitial). Previous research[40] has shown that the monolayer is not affected by surface pressure without protein insertion into the lipid monolayer. Figure
![]() | 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
![]() | 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
The combination of surface pressure–area isotherm and atomic force microscopy allows us to study MBP-incorporated DPPC, DPPS, and DPPC/DPPS monolayers. We can draw these conclusions. (i) The phase behavior of DPPC, DPPS, and DPPC/DPPS in the presence of MBP was significantly different from that of the pure lipid. In addition, the higher
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