Age-related cataracts are the primary cause of vision loss in the elderly population of developing countries. The early detection and prevention of cataracts require increased understanding of the physiological, biochemical, and biophysical bases of lens transparency at the cellular and molecular levels. The goal of this paper was to achieve a greater understanding of the function of Chol in the human eye lens. In this study, we focused our attention on the properties of the phospholipid bilayer saturated with Chol, which models the phospholipid-cholesterol domain, PCD, of the fiber-cell plasma membrane of the eye lens. The molecular-level information of this bilayer cannot be obtained by calorimetric [44
], diffraction [3
], or magic-angle-spinning nuclear magnetic resonance [44
] methods, which have been typically applied to investigate the lateral organization of bulk lens lipid membranes and intact lens membranes. Fortunately, EPR spin-labeling methods enabled us to obtain the missing molecular-level information on the organization and dynamics of lipid molecules in the PCD and also information, how these properties change as a function of the membrane depth [9
]. Those studies showed that in lens lipid membranes as well as POPC bilayers that were saturated with Chol, both oxygen transport parameter and hydrophobicity profiles across the bilayer had a rectangular shape [10
]. In liquid-ordered POPC-Chol bilayers, the rectangular shape of the oxygen transport parameter profile, with an abrupt increase close to the positions of C9 in acyl chains, is observed only for bilayers saturated with Chol (i.e.
, containing 50 mol% Chol). This is in contrast to liquid-ordered POPC-Chol bilayers of the lowest Chol content of ~30 mol% for which the profile is bell-shaped and similar to that for bilayers without Chol. Therefore, it was concluded that saturation with Chol decreases vertical fluctuations of membrane lipids (phospholipids and cholesterol). In effect, the lipids are vertically aligned, and all Chol rings are immersed to the same membrane depth, which is close to the positions of C9 in PC acyl chains. As a result, the membrane surface is smoother compared to membranes without or with lower Chol content.
MD simulations carried out in this study confirm those conclusions. Here, we examined the vertical alignment of selected atoms in POPC and Chol molecules by calculating directly distributions of their positions along the z-axis in the bilayer. The width (Δ) of the distribution for each selected atom is significantly narrowed (from 17-42%) by the presence of a saturating amount of Chol (, and ). MD simulations also confirmed that, indeed, all Chol rings are immersed to the depth close to the positions of C9-C10 in acyl chains as the distribution for C17 () almost overlaps () with that for C210 ().
The difference between ΔN (width for N atoms in the polar headgroups) and ΔC2 () is smaller in the bilayer without Chol than in the bilayer saturated with Chol (). This is an important addition to our earlier experimental results, which showed that the extent of vertical fluctuations of the headgroup, measured as a displacement relative to the glycerol backbone C2 atom, increases after the addition of a saturating amount of Chol [46
], most likely because of a larger available space to the PC headgroups in the bilayer containing Chol [47
], which gives them more motional freedom.
In contrast, the difference between Δs for selected atoms in the PC acyl chains and the Δ for CMs of the POPC molecules is higher in the absence, rather than the presence, of Chol (). This indicates that the local (segmental) motion within acyl chains contributes significantly to the extent of vertical displacements, and that this motion is affected by the presence of Chol. Interestingly, this is more pronounced for the P chain than the O chain (). To elucidate the effect of Chol on segmental motion, analyses of the lifetimes and probabilities of gauche and trans conformations along the acyl chains were carried out. They indicated that, indeed, Chol differently affects torsion angles in the P and O chains. In the P chain, Chol significantly increases the trans conformation lifetime and probability, particularly for even torsion angles but practically does not affect the gauche conformation lifetime ( and ). In effect, the average number of trans/P-chain increases and that of gauche/P-chain decreases (), thus, the P chain is more straight. Tilt-angle analysis additionally showed that the P chain in the POPC-Chol50 bilayer is also more aligned with the bilayer normal than in the POPC bilayer (). All these result in diminished vertical fluctuations of P chain atoms in the POPC-Chol50 bilayer.
The effect of Chol on the O chain is much less straightforward. In contrast to the P chain, Chol has a non-uniform and modest effect on the trans and gauche conformation lifetimes and probabilities in the O chain ( and ). In effect, the average numbers of trans and gauche rotamers per O chain are similar both in POPC and POPC-Chol50 bilayers ().
In light of Chol’s modest effect on the lifetimes and probabilities of trans and gauche rotamers in the O chain, the aligning effect of Chol on the O chain and its upper and lower fragments (δ and ω) is surprisingly high (). In both POPC and POPC-Chol50 bilayers, there is some fraction of the ω segments in which terminal C218 atoms lie closer to the bilayer interface than C210 atoms (tilt > 90°). These fractions are responsible for the bimodal distributions of tilt angles () as well as z-coordinates of C218 (). Thus, the higher values of the Smol profile along the O chain in the POPC-Chol50 bilayer () can be explained by the decreased average tilt of the O chains and their δ and ω segments, as well as the interplay of the probabilities of conformations of torsion angles along the chain. This higher ordering of the O chain results in a narrower distribution of the vertical positions of the chain atoms, but the effect is smaller than in the case of the P chain.
In this study, MD simulations were performed using POPC as a representative phospholipid. However, PC is the major phospholipid in animal lenses, accounting for 35 to 45% of total lipids [14
], but a minor phospholipid in human lenses, accounting for only ~11% of total lipids [14
]. EPR experiments were performed on pig- and cow-lens lipid membranes and, therefore, on lipid bilayers made of POPC, which was the main reason for its usage. Additionally, computer models of POPC membranes are well established and investigated. Recent EPR measurements with sphingomyelin bilayers (the major phospholipid of human lenses [14
]) have confirmed that a saturating amount of Chol also decreases vertical fluctuations in these bilayers [20
The initial regular distribution of POPC and Chol in the bilayer (Fig. S1a, Supplementary Material
) was significantly disturbed after only 100 ns of MD simulation, showing fluctuations of the local Chol concentration (Fig. S1b, Supplementary Material
). However, the average RP (Eq. 2
) changes little with time, particularly after bilayer equilibration (). Therefore, we concluded that local Chol fluctuations do not affect bulk smoothness of the membrane surface. This is supported by the fact that in phosphatidylcholine [21
], sphingomyelin [20
], and lens lipid [9
] membranes saturated with Chol, cholesterol-rich domains do not form and membranes are homogenous on a time scale of 100 ns or longer. Thus, membrane smoothness is not an artifact of the selected distribution of Chol but the property induced by the saturating Chol content.
One of the principal properties of the lens is transparency. Transparency of the eye lens is reduced by light-scattering from different lens elements. Intensive studies have been carried out on this subject and indicate that significant light-scattering may arise from protein density fluctuations [48
] and multilamellar bodies [49
]. The contribution of lens membrane lipids to light-scattering was extensively discussed by Tang et al.
], Michael et al.
], and Bettelheim and Paunovic [53
]. However, light-scattering by the human lens is a very complex physical phenomenon, and contributions from different lens elements are difficult to separate as light-scattering depends on their mutual interaction [50
]. In the case of membranes, light-scattering has its origins in the structural irregularities at the surface and in the bulk of the membrane. Bulk scattering might be described by Rayleigh scattering, as the scattering objects (molecules, density fluctuations) are smaller than the incident light wavelength. Surface scattering arises from random roughness of the membrane surface [56
], described by the average roughness parameter (Eq. 2
). Contribution to surface light-scattering arising from surface irregularities of heights less than 2 nm was measured experimentally in Ref. [57
]. The level of light-scattering at each lipid membrane surface is certainly low. However, in the human lens, the number of cell layers is 1,000 to 2,000. Therefore, light crosses thousands of cell membranes, and scattering is amplified at each surface [56
]. Thus, cholesterol-induced smoothing of the bilayer surface might indeed reduce light-scattering by the lens. Accordingly, based on the present MD simulation results and previous data from EPR spin-labeling measurements, we hypothesize that cholesterol-induced smoothing of the membrane surface should decrease light-scattering and help to maintain lens transparency. This hypothesis will be tested in our future work.
In this article, we have concentrated on the vertical fluctuations of lipid molecules and their atoms in the bilayer. Larger-scale vertical fluctuations of lipid bilayers are thoroughly analyzed in Ref. [58
] but discussion of their contribution to light-scattering by fiber-cell membranes is beyond the scope of this study.
In conclusion, the results of the MD simulations presented here, together with the results of our EPR measurements cited above, have broadened our knowledge and allowed us to better characterize cholesterol-dependent, liquid-ordered membranes saturated with Chol, which have not been investigated extensively thus far and are a major addition to the known condensing effect of Chol [27
]. Our results have also contributed to a better understanding of the function of Chol in the fiber-cell membranes of the human eye lens, especially in consideration of suggestions in the literature that a high amount of Chol in the membrane, together with Chol bilayer domains, helps to maintain lens transparency [3