Different Head Group Dependence on the Lipid Thermodynamic Property of Myelin Basic Protein in the Lipid Monolayer

The interaction between the role of 18.5 KDa myelin basic protein (MBP) isoform and phospholipids has been thought to maintain the stability and compactness of the myelin sheath structure. In this study, we describe the statistical thermodynamic theory of different concentrations’ effects on MBP in the major myelin lipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) ， and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS)) monolayers at the air/subphase interface via Langmuir-Blodgett (LB) technique. A simple statistical mechanical theory is established that predicts the interaction between proteins and phosphate head groups at low surface pressures and the second virial coefficient dependences for the PC, PE, and PS head groups are illustrated. In addition, the surface pressure( π )-mean molecular area(mma) curves were also analyzed using two-dimensional virial equation of state (2D-VES). The positively charged showed that MBP may integrate into different lipid monolayers via hydrophobic and electrostatic interactions, which was found to be consistent with AFM observations of domain and aggregate structures as well as with changes in the surface morphology induced by MBP. These analyses pertaining to membrane structure will provide better insight into membrane modeling systems, especially the interaction between membrane molecules.


Introduction
Myelin basic protein (MBP) is an important component of myelin. The severity and extent of nerve injury as well as its outcome and prognosis of the disease require the detection of MBP concentration in serum and cerebrospinal fluid (CSF). The central nervous system (CNS) and peripheral nervous system (PNS) compacted myelin proteins are MBP, which accounts for about 30% of the total myelin sheath [1,2]. Specifically, 18.5 KDa MBP is the main protein in the mature myelin of the CNS and is the most conserved protein in the MBP family during evolution. It is reported that the 18.5 KDa MBP of vertebrates contains 170 amino acid residues that play a key role in the structure and function of the protein [3,4]. It interacts with the phospholipids of different heads of the myelin membrane, promoting the fusion of oligodendrocytes and forming the main dense line of the multilayer membrane structure [5,6]. For good measure, the interaction between MBP and myelin membrane phospholipids may be closely combined, which confers stability to the myelin structure and function. Moreover, nerve conduction insulation is improved along with conduction speeds, playing a very important role in myelin formation as well as brain differentiation and maturation [7][8][9].
In our previous studies [10][11][12], we have investigated the adsorption of MBP at the free air/subphase interface and onto different monolayers at the interface. The results showed that the surface phenomena are closely related to the concentration of protein, surface pressure, subphase and so forth. Additionally, we found that the adsorption of MBP into the various phospholipids was necessary due to the electrostatic and hydrophobic interactions between the penetrating MBP and the negatively charged head and hydrocarbon chains of the phospholipids. However, other studies have shown that the lipid monolayers possessed a lipid composition of myelin, such as dipalmitoylphosphatidylcholines (DPPC) [13], dipalmitoylphosphatidylserine (DPPS) [14], phosphatidylglycerol (PG) [15], and phosphatidicacid (PA) [16]. Among them, the myelin lipids (neutral POPC and POPE as well as anionic POPS) had interesting thermodynamic properties in the two-dimensional interfacial phase. POPC, POPE, and POPS have the same hydrophobic tail chain and contain 18 carbon atoms in each fatty acyl chain, but their hydrophilic headgroups are different [17].
The chemical structures of MBP, POPC, POPE, and POPS are shown in Fig.1.
This investigation does not study the transverse structure of myelin such as nerve impulse propagation, conductivity, and membrane order perturbation; it focuses on the lateral organization adopted by the proteins and myelin lipids when the monolayer is formed at the air/subphase interface.
Essentially, the influence of MBP on the dynamics and structure of myelin membranes model was explored using Langmuir-Blodgett (LB) technology and an atomic force microscope (AFM). AFM is a powerful tool for the analysis of lipid-protein interactions and the formation of microdomains. This study regarding the supramolecular structure of myelin is of biophysical significance and medical value.

Chemicals
The protein used in the experiments, MBP, was collected and extracted from the bovine brain and purified in a water-soluble solution according to the procedures described by Deibler et al. [18]. It was then dialyzed against pure Millipore water and used at a concentration of 1.0×10 -9 M. The buffer was 10 mM Tris-HCL, and the pH was adjusted to 7.2. 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC, molecular weight 760.1 g/mol), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (sodium salt) (POPE, molecular weight 718.0 g/mol), and 1-palmitoyl-2-oleoyl-sn-glycero-3phospho-L-serine (sodium salt) (POPS, molecular weight 784.0 g/mol) of high a purity (at least 99%) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA) and used without further purification. Working solutions of lipids (1mg/ml) were dissolved in chloroform/methanol 3:1 v/v mixture. The water (with a resistivity higher than 18.2 MΩcm) used in the experiment was obtained from a Millipore purification system.

Isotherms
Langmuir experiments were fabricated with a KSV Minitrough system (Helsinki, Finland) with an operational area of 273 cm 2 . All experiments were performed using Tris-HCl as the subphase. The Tris-HCl buffer solutions were prepared by Tris (hydroxyethyl) amino-methane (concentration of 10 mM) and titrated with HCl solution to the desired pH value (pH = 7.2). Prior to each measurement, the LB trough and barriers were thoroughly washed and wiped with ethanol and triple distilled water several times. The appropriate amount of lipid solutions (30 µL) was spread with precision microvolume syringes onto the subphase containing the moderate MBP. The solvent was allowed to evaporate for 15 minutes until the surface pressure (π) was stabilized. Subsequently, the monolayer was compressed to obtain the surface pressure versus area (π-A) isotherms. The constant rate of barrier during compression was 10 mm/min at a fixed temperature of 22±1 ℃ via circulating water bath.
Each run was repeated at least three times to obtain reproducible results.

Langmuir-Blodgett (LB) transfer
Transfer of amphiphilic monolayers onto the mica substrate was achieved using the Langmuir-Blodgett technique. Prior to transferring, freshly cleaved mica was immersed into the Tris-HCl subphase. Then, the barriers were compressed up to the desired surface pressure of 10 mN/m. Following 15 minutes of stabilizing the monolayer, the mica sheet was removed from the subphase with a constant dipper rate of 20 mm/min to obtain the transfer ratio of 1.0 ± 0.1.

Atomic force microscopy
The transferred amphiphilic monolayers were monitored using a SPM-9500-J3 Atomic Force Microscope (AFM, Shimadzu Corporation, Japan). The scans were performed in contact mode using a Micro V-shaped Cantilever (Olympus Optical Co. Ltd., Japan) with a spring constant = 0.06 N/m, length of 100 μm, and thickness of 400 nm. The scan rate was 1.0 Hz per line with a resolution of 512 pixels per line.

Phase transitions
The viscoelastic properties of the different monolayers were determined by calculating surface compressibility modulus values ( 1 s C − ) from the π-A curves as: where A is the average molecular area at the indicated surface pressure and π is the corresponding surface pressure. The higher the compression modulus value of 1 s C − , the lower the elasticity of the monolayer, or the more ordered the monolayers [19].

Virial coefficients for different lipids and MBP interactions
The π-A isotherms in the region starting from the isotherms to π = 10 mN/m were fitted into the following two-dimensional virial equation of state (2D-VES) [20]: where a denotes the aggregation coefficient. If a = 1, there is no aggregation between molecules at a low surface pressure, however, when a < 1, aggregation occurs between molecules, and the lower the value of a, the higher the degree of aggregation. b is the virial coefficient, where b > 0 and b < 0 characterize the repulsive and attractive properties of the intermolecular interaction, respectively.

Results and discussion
π-A isotherm measurements of Tris-HCl subphase containing 1nM MBP with POPC,  (Fig.2b). In terms of POPS, the isotherm demonstrated a LE phase state of the monolayer. The collapse pressure of the isotherm appears at π = 36.5 mN/m [25]. the isotherms with 1nM MBP show higher mma's than the isotherm of pure POPS. This outcome was probably due to the adsorption of MBP onto the monolayer.

Two-dimensional virial equation of state for different lipids and MBP interactions
At a low surface pressure (10 mN/m), in order to better explore the characteristics of MBP with phospholipids of different head groups, the π-A curves were analyzed using simple statistical theory and depended on the lipid surface roughness, as seen in Fig.7(b, c). These results are in accordance with the previous analysis of the second virial coefficients, which further illustrates that MBP-lipid (POPC, POPE, and POPS) interactions show a significant dependence on concentration and is likely governed by electrostatic and hydrophobic forces. Such interactions are further illustrated by the model in Fig.8.

Conclusions
In conclusion, this study demonstrates the analysis of the monolayers' characteristics, The presence of MBP induced conformational changes in the monolayer when mixed with three lipids.
When mixed with POPE+MBP and POPS+MBP, the MBP particles were adsorbed into the monolayer.
In terms of POPC, its molecules were squeezed into the subphase. (3) These findings may provide qualitative and quantitative information on MBP adsorption mechanisms for major myelin lipids (POPC, POPE, and POPS), which maintain the tightly packed multilamellar structure of the myelin sheath by hydrophobic and electrostatic interactions.
The results of our study to rise the questions about interactions of MBP with other lipid models, such as "lipid raft", "plasma membrane", and "myelin membrane model". The myelin lipid bilayers had a lipid conformational characteristic of myelin from "healthy" and "disease-like" in the cytoplasmic leaflets. They exhibit different adsorption mechanisms. We will study the relevant issues in this field in due course.