Structural Change in Dimyristoyl Phosphatidyl Choline (DMPC) Molecule Alkyl Chains on the Addition of Cholesterol to DMPC Monolayer: Specific Addition Effect of Cholesterol

We have investigated the morphology of dimyristoyl phosphatidyl choline (DMPC)—cholesterol mixed monolayer formed on the water surface by dropping method using surface tension measurement (STm), Brewster angle microscopy (BAM), and infrared external reflection spectroscopy (IERS). STm results showed negative deviation of the limiting molecular area (A 0 ) of cholesterol occurred when cholesterol was added to the DMPC monolayer. BAM images showed the expandable DMPC monolayer changed to the condensed rigid monolayer at more than cholesterol mole fraction (x Chol ) 0.4. IERS re-cordings showed that the addition of cholesterol at x Chol = 0.4 occurred structural change from gauche- to trans- conformation of two DMPC molecule alkyl chains. From these results, it is found that cholesterol molecule has specific properties that cause structural transition of DMPC molecule alkyl chains.


Introduction
The monolayers are formed on the water surface when amphiphilic substances biomembrane. Therefore the lipid monolayer is selected as a biomembrane model to investigate cellular functions such as membrane structure, interaction with functional substances, molecular recognition, and transmission of functional materials in and out of the living biological cell membrane [1] [2] [3] [4].
Cholesterol (Figure 1(a)) is classified under the steroid group. Structurally, it is composed of a steroid skeleton and a hydrophilic hydroxyl group (-OH).
Cholesterol is one of the main components in biomembrane and the important organic compound from the viewpoint of various biological phenomena such as controlling of biomembrane conformation, functioning of integral biomembrane proteins, and metabolism in the living body. In these phenomena, "condensation effect" of cholesterol is widely known. The addition of cholesterol in biomembrane alters the order of biomembrane structure and affects the fluidity of the biomembrane. This also promotes thermal and structural stabilities of biomembrane in a wide temperature range. Many studies on the condensation effect have been performed using phospholipid-cholesterol mixtures in monolayer and bilayer systems, and liposomes [2] [5]- [11]. The techniques of thermal analysis, ATR-FTIR and NMR spectroscopy, and molecular simulation [5] [12] [13] [14] [15] [16] showed that the structure of DMPC molecule exchanged from liquid-disordered (L d ) phase to liquid-ordered (L o ) phase with an increase in cholesterol mole fraction.
In this communication, we have reported the cholesterol-mixed effect on the morphology of expandable dimyristoyl phosphatidylcholine (DMPC) (Figure 1

Monolayer Formation
DMPC and cholesterol were dissolved in chloroform to prepare dropping solution. The solution was dropped on a purified water surface with a 100 μl microsyringe (Ge-0583-04; Hamilton Corp., Nevada, USA) to form monolayer. The details of the method of monolayer preparation have been reported in a previously publication [17]. 1 μl drop of the solution was dropped gently on the water surface and the next drop was added after ≥1 min so that previous droplet expanded sufficiently on the water surface. The arrival of equilibrium by proper expanding of DMPC and cholesterol on the surface was confirmed by the constant surface tension value attainment after the addition of each droplet (Section 2.3.). The process of the solution addition was repeated until completion of monolayer formation was done. The completion of the monolayer was confirmed by observing the formation of lenses on the water surface and the value of surface tension (Section 2.3.) did not change any more.

Surface Tension Measurement (STm)
Surface tension measurement (STm) for each mixed monolayer was carried out on a Surface Tensiometer (CBVP-A3; Kyowa Interface Science Corp. Ltd., Saitama, Japan) with a platinum Wilhelmy plate. Surface tension was recorded at each dropping at 26˚C ± 1˚C. The surface tension values were then converted into surface pressure (π). The π-A isotherm curves for each monolayer were constructed as a function of the dropping volume of the monolayer forming components in terms of molecule numbers.

Brewster Angle Microscopy (BAM)
Brewster angle microscopy (BAM) was employed for visualizing the morphologies of each DMPC-cholesterol mixed monolayer using a commercial BAM microscope (EMM633K; Filgen Inc., Nagoya, Japan) attached with an USB-CAP Journal of Biophysical Chemistry type (SD-USB2CUP3; AREA Co. Ltd., Tokyo, Japan) image analysis software.
BAM records the monolayer images arise from the refractive index difference between the monolayer and the water phase. BAM device was mounted on a glass dish and p-polarized light of 632.8 nm wavelength from a 10 mW He-Ne laser was irradiated at the Brewster angle of 53.1˚ on each mixed monolayer surface. The reflected light was magnified with a 40 mm focal length lens and detected by a CCD camera (C5948-70; Hamamatsu Photonics, Hamamatsu).
The formation process of each mixed monolayer by dropping method was observed on real-time at 26˚C ± 1˚C. The lateral resolution of BAM images was about 1 μm.

Infrared External Reflection Spectroscopy (IERS)
The structure of DMPC molecule alkyl chains in DMPC monolayer and DMPCcholesterol mixed monolayer was investigated by infrared external reflection spectroscopy (IERS). The details of the method of IERS measurement have been reported in a previously publication [18]. An   The DMPC monolayer π-A isotherm curve (○) without cholesterol is the same as reported previously [19] [20]. The π value increased gradually at about 1.2 nm 2 of A and showed a monotonous increase up to 43 mN/m. The shape of the curve was smooth and similar to that obtained by compression method [21] [22] [23]. The limiting molecular A 0 was 0.74 ± 0.02 nm 2 /molecule corresponded to 40 mN/m of π and the value was very similar (0.73 nm 2 /molecule) obtained at 40 mN/m in compression method. From the pattern of π-A isotherm curve, it was confirmed that DMPC monolayer by dropping method formed liquid expanded (LE) state where two DMPC molecule's alkyl chains were disordered partially.

Surface Tension Measurement (STm)
The structure of alkyl chains contained gauche conformation, thereby the crosssectional area would be larger than of the hydrophilic group. For the cholesterol monolayer, the π-A isotherm curve (•) in Figure 2 showed that the π increased a little gradually and subsequently sudden steeply up to 30 mN/m. A 0 was 0.39 ± 0.02 nm 2 /molecule at 30 mN/m of π and this value was also very similar (0.40 nm 2 /molecule at 35 mN/m) in compression method [22] [23] [24] [25]. The obtained A 0 was also identical (0.41 nm 2 /molecule at 34 mN/m) as reported previously by dropping method [11].
A 0 of DMPC-cholesterol mixed monolayer in this research was compared to that of the ideal mixed monolayer that is calculated from each A 0 of DMPC and cholesterol monolayers.     (Figures 4(q)-(t)) was almost the same as those of reported previously by compression method [29] [30] [31]. Therefore for condensable molecule such as cholesterol, the condensed monolayer is formed through the same aggregation process on the water surface either by dropping or compression methods.    Figure 5(c) is the absorption spectra (k-spectra) obtained from simulation calculation based on the solid experimental line in Figure 5(a) and Figure 5(b) [18]. In Figure 5(c), the vertical axis represents imaginary part of the complex refractive index (k), solid line (-) spectra (k xy ) is of the components parallel to the surface plane (in plane), dotted line (---) spectra (k z ) is of the components perpendicular to the surface plane (out of plane) [18].

Infrared External Reflection Spectroscopy (IERS)
As shown in the k-spectra of Figure 5(c) (k z ), five absorption peaks were assumed in simulation at 2958, 2920, 2897, 2872 and 2852 cm −1 . These peaks were assigned to CH 3 anti-symmetric stretching vibration band ν as (CH 3 ), CH 2 anti-symmetric stretching vibration band ν as (CH 2 ), Fermi resonance CH 3 symmetric stretching vibration band ν s (CH 3 ), and CH 2 symmetric stretching vibration band ν s (CH 2 ), respectively. The two peaks at 2920 and 2852 cm −1 were strong in k xy spectrum assigned to the methylene groups in the two DMPC molecule alkyl chains.
To investigate the form of the two alkyl chains (the hydrophobic group) in the monolayer, we focused on the information of two stretching vibration bands (ν as (CH 2 ) and ν s (CH 2 )) of methylene groups (-CH 2 -) in Figure 5(c). The bands (ν as (CH 2 ) and ν s (CH 2 )) appeared at 2920 and 2852 cm −1 , respectively are characteristics of gauche conformation of alkyl chain [32] [33]. Therefore the hydrophobic DMPC molecule alkyl chains in the monolayer are in an irregularly kinked form. solid line (-) shows the absorption spectrum (k-spectrum) obtained from the simulation calculation [18] conducted using the solid line (-), the experimental IER recording shown in Figure 6(a). The solid lines (-) of Figure 6(b) is the absorption spectrum (k-spectra) (k xy ) obtained from simulation calculation based on the solid experimental line in Figure 6(a) [18]. For a comparison, the (k xy ) of DMPC monolayer in Figure 5(c) is also described as the dotted lines (---) in Figure 6(b).
The two strong absorption peaks ν as (CH 2 ) and ν s (CH 2 ) were optimized to fit the experimental IER spectrum, resulting the peak position of k xy (solid lines (-)) at 2919 cm −1 and 2849 cm −1 , respectively, for DMPC-cholesterol (x Chol = 0.4) mixed monolayer. These peaks were assigned to CH 2 anti-symmetric stretching vibration band ν as (CH 2 ) and CH 2 symmetric stretching vibration band ν s (CH 2 ), respectively.  Comparing the two k-spectra of DMPC monolayer with (-) and without cholesterol (---) in Figure 6(b), two differences in peak position and absorption intensity were noticed. The peak positions of DMPC-cholesterol spectrum (-) ν as (CH 2 ) and ν s (CH 2 ) at 2919 and 2849 cm −1 , respectively, were at shorter wavelengths than DMPC spectrum (---) ν as (CH 2 ) and ν s (CH 2 ) at 2920 and 2852 cm −1 , respectively. The absorbance intensities of the former mixed monolayer's bands were 1.3 times higher than the latter. Snyder and Strauss [32] reported in C-C bond of polymethylene a lower (blue shift) in two ν as (CH 2 ) and ν s (CH 2 ) stretching vibration bands resulted due to change from gaucheto transconformation. Therefore the blue shift is suggested that the kinked form (gauche conformation) of DMPC molecules decreased by the addition of cholesterol to the monolayer. Thus addition of cholesterol caused the structural transition from gaucheto transconformation. The increase in the absorption intensity of the two peaks (ν as (CH 2 ) and ν s (CH 2 )) by the addition of cholesterol also supports the structural change described above. The trans conformation consecutively arranged in the alkyl chain in all-trans zig-zag form with the long axis along the surface normal with parallel to the -CH 2 -plane at the surface plane. This arrangement makes the peak intensities of ν as (CH 2 ) and ν s (CH 2 ) higher ( Figure 6(b)) on the addition of cholesterol in the k-spectra (in k xy plane) [18].
The blue shifted stretching frequency of ν as (CH 2 ) and ν s (CH 2 ) vibration bands of the two DMPC molecule alkyl hydrophobic chains, with the addition of cholesterol in the DMPC monolayer, leads the mixed monolayer more ordered and condensed by a decrease in the area of those alkyl chains with the structural change from disorder to order. This also corresponds to the negative deviation from the ideal line in Figure 3. The structural transition from gaucheto transconformation due to the addition of cholesterol occurred in the DMPC molecule Journal of Biophysical Chemistry alkyl chains. The alkyl chains ordered structure and more condensed state also corresponded to the STm result in Figure 2 that the shape of π-A isotherms curve showed LC state close to that of cholesterol.

Conclusions
In The results obtained in this research showed first the ordering of DMPC molecule occurred even at the level of monolayer on the water surface and the ordering was related not only to the general condensation effect of cholesterol but also to the gaucheto transconformation change in the two DMPC molecule alkyl chains. The ordering might have induced changes in the density and orientation of hydrophilic group of DMPC molecule at monolayer-water interface. More experiments are needed to obtain further information about the effect of cholesterol on the DMPC molecules in the DMPC-cholesterol mixed monolayer, using several other physicochemical methods such as surface potential measurement and vibration sum frequency generation (VFSG) spectroscopy.