Cholesterol-Induced Structural Changes in Saturated Phospholipid Model Membranes Revealed through X‑ray Scattering Technique Rajendra P. Giri, Abhijit Chakrabarti, and Mrinmay K. Mukhopadhyay* Saha Institute of Nuclear Physics, HBNI, 1/AF, Bidhannagar, Kolkata 700064, India ABSTRACT: Lateral and out-of-plane organization of cholesterol and its effect on regulating the physicochemical properties of zwitterionic phospholipid model membranes have been investigated by a pressure− area isotherm study from the Langmuir monolayer, atomic force microscopy (AFM), and X-ray reflectivity (XRR) measurements from supported binary monolayer films. The systematic isotherm studies on the Langmuir monolayer of phospholipids and the subsequent extraction of excess Gibbs free energy (ΔG exc ) revealed the mechanism of cholesterol interaction and the molecular cooperativeness for di fferent arrangements in the phospholipid model membranes. We have found a critical cholesterol molar concentration (χ c ) up to which the lipid−cholesterol miscibility gradually increases and then further increase in the concentration leads to an inhomogeneous structure formation similar to raft structures. The thickening in the lipid acyl chain and the subsequent lowering of the lipid head group thickness up to χ c are also evident from the XRR study. Beyond χ c , large-sized domains are observed in the AFM images from the deposited monolayer. χ c has also been observed to depend on the phase of the monolayer, in particular, ∼25 molar % in the gel phase and ∼40 molar % in the fluid phase, wherein a regular distribution has been found with the highest separation between the cholesterol molecules. The extracted isothermal compressibility coefficient (C S ) and ΔG exc from the monolayer isotherms indicate that the molecular arrangement at χ c are the most stable configurations of the monolayer. Our study provides direct evidence into cholesterol-induced evolution in phase behavior and the consequent model on the structure at different phases in the phospholipid Langmuir monolayers. ■ INTRODUCTION The phase behavior and structural properties of the model biomembranes are of considerable scientific and practical interest as they provide a basic platform for understanding the membrane functions in living cells. 1,2 The fluid mosaic model 3 that treats the biomembranes as a homogeneous mixture of cellular compo- nents has been seen no longer to be crudely valid in the eukaryotic cell membranes. The modern concept of the cell membrane structure envisions the presence of an ordered structure of cholesterol-enriched micro-domain assemblies, known as lipid rafts, in a relatively disordered lipid matrix. 4,5 These assemblies are believed to be involved in a number of cellular processes, including drug delivery, protein sorting, signal transduction, and so forth. 1,2,6 Cholesterol, an amphipathic molecule very often treated as a rigid cylinder, is an abundant class of lipids regulating the physicochemical properties of the mammalian cell membranes. 7 Even a small cholesterol fraction in the model lung surfactant can offer control over the surfactant spreadability by reducing the viscosity of the surfactant interface by orders of magnitude. 8 In addition, the phospholipid acyl chains, in the physiologically relevant liquid crystalline state, show a higher degree of orientational and conformational order in the presence of cholesterol. 9,10 It is believed to have a strong effect on the structure and functions of proteins residing in the membrane. The chemical structure of a cholesterol molecule includes a tetracyclic fused ring in a trans-configuration along with a flexible hydrocarbon side chain that helps to closely attract the phospholipid acyl chains toward it with van der Waals forces acting between them. This fact leads to a laterally more condensed membrane with a higher packing density of phospholipids making it less permeable. Thus, cholesterol affects the conformational order and membrane permeability and regulates the lateral organization of the membrane compo- nents. 11−19 There are some existing mechanical models, namely, the condensed complex model, 20,21 the super lattice model, 22,23 and the umbrella model, 9,23 which are being used to explain the physical mechanism of the phospholipid−cholesterol interac- tion. 24 It was reported earlier 25 that the miscibility of cholesterol increases significantly in the saturated phosphatidylcholine (PC) lipids in comparison to that in the unsaturated one. Previous reports concerning the PC−cholesterol interaction both in the Langmuir monolayer 26−28 and in the model bilayer 22,24,29−32 have witnessed the cholesterol-induced structural changes in the phospholipid model membranes. Despite vivid research with model biomembranes, the phospholipid phase dependency of lateral and out-of-plane organization of cholesterols as a function Received: December 14, 2016 Revised: March 14, 2017 Published: April 6, 2017 Article pubs.acs.org/JPCB © 2017 American Chemical Society 4081 DOI: 10.1021/acs.jpcb.6b12587 J. Phys. Chem. B 2017, 121, 4081−4090