Article pubs.acs.org/Langmuir
Crucial Role of the Double Bond Isomerism in the Steroid B‑Ring on the Membrane Properties of Sterols. Grazing Incidence X‑Ray Diffraction and Brewster Angle Microscopy Studies Michał Flasiński,*,† Paweł Wydro,‡ Marcin Broniatowski,† Katarzyna Hąc-Wydro,† and Philippe Fontaine§ †
Department of Environmental Chemistry, Faculty of Chemistry, Jagiellonian University, Gronostajowa 3, 30-387, Kraków, Poland Department of Physical Chemistry and Electrochemistry, Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060, Kraków, Poland § Synchrotron Soleil, L’Orme des Merisiers, Saint Aubin, BP 48, 91192 Gif-sur-Yvette Cedex, France ‡
S Supporting Information *
ABSTRACT: Three cholesterol precursorsdesmosterol, zymosterol, and lanosterolwere comprehensively characterized in monolayers formed at the air/water interface. The studies were based on registration of the surface pressure (π)−area (A) isotherms complemented with in situ analysis performed with application of modern physicochemical techniques: grazing incidence X-ray diffraction (GIXD) and Brewster angle microscopy (BAM). In this approach we were interested in the correlation between molecular structures of the studied sterols found in the cholesterol biosynthetic pathway and their membrane properties. Our results revealed that only desmosterol behaves in Langmuir monolayers comparably to cholesterol, the molecules of which arrange in the monolayers into a hexagonal lattice, while the two remaining sterols possess extremely different properties. We found that molecules of both zymosterol and lanosterol are organized on the water surface in the two-dimensional oblique unit cells despite the fact that they are oriented perpendicular to the monolayer plane. The comparison of chemical structures of the investigated sterols leads to the conclusion that the only structural motive that can be responsible for such unusual behavior is the double bond in the B sterol ring, which is located in desmosterol in a different position from in the other two sterols. This issue, which was neglected in the scientific literature, seems to have crucial importance for sterol activity in biomembranes. We showed that this structural modification in sterol molecules is directly responsible for their adaptation to proper functioning in biomembranes.
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INTRODUCTION Sterols comprise a broad class of steroid lipids possessing in their molecular structure a characteristic system of four fused cycloalkane rings, three of which (A, B, and C) are six-member cyclohexanes and one (D) is a five-member cyclopentane.1 Since sterols are direct derivatives of squalene (C30), made up of six C5 isoprenoid building blocks, these compounds are also classified as triterpenoids.2 In sterol molecules the fully saturated cyclohexane rings are in a chair conformation resulting in all-trans, nonplanar structure of the entire molecule of sterols like dihydrocholesterol or coprostanol. However, in most sterols including cholesterol, at least one double bond is present in ring B. The presence of this bond influences the whole structure by the special changes of the B-ring, which acquires the conformation of half-chair, boat, or twisted boat conformation, depending on the double bond location.2−4 Apart from the characteristic steroid rings, the molecules of sterols have aliphatic, typically eight-carbon-atom chains attached to the C17 carbon atom of the cyclopentane ring which can be fully hydrogenated, possess one or more double bounds, and even possess additional substituents. The C3 © 2015 American Chemical Society
hydroxyl group, by contrast, provides the amphipathic character to sterol molecules enabling them to be significant components of biological lipid membranes.5,6 The class of sterols as well as their distribution in the living organisms depends on the particular species and even on the specification and function of a tissue and the cell type.4,7,8 In this work we are focused on the comparative characteristics of three selected sterols, differing as regards molecular structures: desmosterol, zymosterol, and lanosterol. These compounds are known to be important precursors in the biosynthesis of cholesterol.9 Lanosterol is the first sterol intermediate formed by the conversion of isoprenoid squalene, catalyzed by the enzymes epoxidase and cyclase.10,11 In the subsequent step, series of demethylation reactions occur resulting in formation of zymosterol lacking, as compared to lanosterol, three methyl groups at the C14 and C4 positions.2 The chemical structures of the investigated sterols are presented in Scheme 1. Received: March 10, 2015 Revised: June 9, 2015 Published: June 10, 2015 7364
DOI: 10.1021/acs.langmuir.5b00896 Langmuir 2015, 31, 7364−7373
Article
Langmuir
Scheme 1. Chemical Structures of the Investigated Representatives of Sterols Family: (a) Cholesterol, (b) Desmosterol, (c) Zymosterol, and (d) Lanosterol
nevertheless, the understanding of their behavior at the air/ water interface is an essential starting point for such a discussion. In our studies we applied the Langmuir monolayer technique in order to have control over the surface parameters of the investigated sterol monolayers, e.g., the surface density of the monolayers, together with the complementary techniques: BAM and GIXD. Such an approach allowed us to achieve essential conclusions regarding the properties of the investigated sterols in 2D molecular layers and emphasize the main differences between these compounds.
All these molecules have a backbone built from four cycloalkane rings and identical aliphatic tails monounsaturated at the C24 carbon atom. The structural differences between these compounds concern the position of the double bond in the ring system as well as the number of methyl groups. In the molecule of desmosterol, similarly to cholesterol, the unsaturated bond is located between C5 and C6 carbon atoms (Δ5 bond), whereas in zymosterol and lanosterol it is in the Δ8 position. Furthermore, lanosterol differs from the two remaining sterols as regards the three extra methyls: two attached to the C4 carbon atom and one attached to the C14. This difference has significant influence on the steric properties of the lanosterol molecule. Namely, the α-face of this compound is not as smooth as in the other sterols, including cholesterol, but it is substituted by additional methyl groups which provides increased roughness at this site.1,12 The structural differences in the sterols produce shape changes that affect the interactions and reactivity potential with other components in the membrane.13 These interactions are crucial from the point of view of the sterol functions in biomembranes which are connected to the regulation of such physicochemical parameters as fluidity,14 stiffness,15 permeability,16,17 and molecular ordering.18 In the present comprehensive studies we are interested in the film-forming properties of the three cholesterol intermediates. It is worth mentioning that the knowledge regarding the behavior of these sterols in monomolecular films can be correlated with the properties and potential activity of these molecules in natural lipid bilayers. A good example of the correlation between results obtained in the simple two-dimensional (2D) model with the functioning of molecules in natural biological membranes concerns cholesterol. The main properties of this sterol have been studied with application of the Langmuir monolayer technique complemented by modern physicochemical techniques, like Brewster angle microscopy (BAM) or reflection spectroscopy (polarization modulation infrared reflection absorption spectroscopy, PM-IRRAS) or methods based on scattering of X-ray synchrotron radiation (grazing incidence Xray diffraction (GIXD) and XR). This concerns, e.g., the wellknown condensing and ordering effect of cholesterol on acyl chains of membrane phospholipids19−21 as well as the properties connected to the capability of lipid domain formation.22,23 Unfortunately, apart from cholesterol, the studies on the other membrane sterols are not comprehensive in nature. The investigations on the activity of sterols in lipid membranes need obviously the studies concerning their interactions with other classes of membrane components;
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EXPERIMENTAL SECTION
Materials. Sterols applied in our investigations were synthetic products of the highest purity available in the suppliers: ≥99%. Desmosterol, zymosterol, and lanosterol were purchased from Avanti Polar Lipids Inc., USA. In each experiment 100−200 μL of ca. 0.25 mg/mL sterol solution in chloroform/methanol 9/1 (v/v) mixture was deposited onto water subphase with a Hamilton microsyringe, precise to ±1.0 μL. Applied solvents chloroform of spectroscopic purity (99.9% stabilized by ethanol) as well as methanol (99%) were purchased from Sigma-Aldrich. As a subphase ultrapure water of resistivity ≥18.2 MΩ·cm−1 obtained from a Milli-Q system was applied. To ensure the best reproducibility of the registered isotherms, all measurements were repeated at least three times. Methods. The initial experiments were performed with a NIMA (U.K.) Langmuir trough with a total area of 300 cm2, placed on an antivibration table. The surface pressure was measured with an accuracy of ±0.1 mN/m using a Wilhelmy plate made of filter paper (ashless Whatman Chr1) connected to an electrobalance. In a routine experiment, the monolayer was left to equilibrate for at least 5 min before the monolayer compression was started with a barrier speed of 20 cm2·min−1. The constant temperature during experiments (20 °C) was controlled thermostatically with the circulating water system. Brewster Angle Microscopy. BAM experiments were performed with an UltraBAM instrument (Accurion GmbH, Gö ttingen, Germany) equipped with a 50 mW laser emitting light of p polarization at a wavelength of 658 nm, a 10× magnification objective, a polarizer, an analyzer, and a CCD camera. The spatial resolution of the BAM was 2 μm. The microscope was installed over the KSV Langmuir trough with a total area of 700 cm2 equipped with two movable barriers enabling symmetrical compression. Each experiment based on application of BAM was repeated three times in order to obtain the best representation of the images. Grazing Incidence X-ray Diffraction (GIXD). GIXD experiments were carried out at the SOLEIL synchrotron center (Paris, France) on the liquid surface diffractometer at the SIRIUS beamline. The dedicated Langmuir trough placed in a gastight canister is mounted on the goniometer of the diffractometer. Before the experiment, the canister is sealed and flushed with helium to reduce the oxygen level in order to reduce the scattering background and to minimize the beam damage during the experiment. After at least 30 min a monolayer was 7365
DOI: 10.1021/acs.langmuir.5b00896 Langmuir 2015, 31, 7364−7373
Article
Langmuir compressed to the target surface pressure (5 or 30 mN/m), which was held constant during the entire experiment. The surface pressure was measured with a Wilhelmy balance (R&K, Germany) equipped with a filter paper strip as a π sensor. The detailed construction of the diffractometer working at the SIRIUS beamline and the parameters of the synchrotron beam applied in the GIXD experiments are described at the SOLEIL Web site (www.synchrotron-soleil.fr) as well as in the previous paper.24 The application of the GIXD technique allows attainment of high resolution information about the in-plane organization of film-forming molecules on the angstrom scale. Diffraction of X-rays in the technique is possible only if the molecules are periodically organized at the interface. If the incidence X-ray radiation is scattered, its intensity can be measured by a position sensitive detector (PSD). The scattered intensity is measured by scanning over a range of horizontal scattering vectors Qxy, defined as
Q xy ≈
⎛ 2θxy ⎞ 4π sin⎜ ⎟ λ ⎝ 2 ⎠
where 2θxy is the angle between incident and diffracted beams projected onto the horizontal plane. Bragg peaks are resolved in the Qxy direction, by integrating the scattered intensity over Qz. Lattice vectors a and b can be correlated with the position of the Bragg peaks’ maxima according to the relation
Figure 1. Surface pressure (π)−mean molecular area (A) isotherms registered for monolayers of four investigated sterols: cholesterol, desmosterol, zymosterol, and lanosterol.
⎡ h2 ⎤−1/2 ⎛ hk ⎞ 2π k2 ⎜ ⎟ d= cos γ ⎥ sin λ =⎢ 2 + 2 −2 ⎝ ab ⎠ Q xy ⎣a ⎦ b
smaller as compared to the monolayers of desmosterol and zymosterol. Another difference concerns the collapse of the investigated monolayers. In the case of the black, red, and blue curves in Figure 1, at π = 41, 44, and 41 mN/m, respectively, the surface pressure stops to rise further, indicating collapse of the surface film. This feature is characteristic for these monolayers at the area/molecule of ∼37 Å2, which is in accordance with the cross-sectional area of sterol molecules (e.g., cholesterol).28 On the other hand, the course of the curve obtained for the monolayer of lanosterol, at high surface pressures, is different; however, the analysis of the isotherm leads to the conclusion that the collapse of the monolayer occurs at a very similar molecular area. The difference in characteristics of the studied monolayers as well as the information regarding the monolayer state can be confirmed on the basis of Cs−1(π) dependency (inset in Figure 1). According to the definition, the compression modulus, expressed as Cs−1 = −A(dπ/dA), was calculated from the surface pressure (π)−mean area (A) isotherm. It can be seen that the monolayers of cholesterol, desmosterol, and zymosterol reach a high degree of condensation, which is manifested in large compression modulus values exceeding 700 mN/m. From the point of view of traditional interpretation of the monolayer state proposed by Davies and Rideal,29 such high values of Cs−1 (>250 mN/m) are characteristic for monolayers being in the solid state (S), in which well-ordered molecules in the surface film are oriented perpendicular to the air/water interface plane. On the other hand, in the case of lanosterol monolayer, in a wide range of the surface pressure, the values of the compression modulus reach ca. 150 mN/m, which is characteristic for the liquid condensed state (LC) containing closely packed molecules tilted from the surface normal. Such an interpretation, however, is less probable taking into account the chemical structure of the studied compound. Nevertheless, in order to confirm this conclusion, the information obtained from complementary techniques is required. It is also worth mentioning that both the course and the shape of the π−A isotherm registered for the surface film of lanosterol is reminiscent of the compression curves typical for
where d is the lattice repeat distance in the 2D lattice, h and k are Miller indices, and γ is the angle between lattice vectors of the given unit cell. For the hexagonal unit cell, γ = 120°, for the rectangular cell γ = 90°, and for the oblique cell, the angle is
