The results presented here demonstrate that the CBD, which is formed in a lipid suspension when the cholesterol content exceeds the CST, has pronounced effects on the organization and dynamics of phospholipids in the PCD. The CBD increases the order of phospholipid alkyl chains and decreases their dynamics. The presence of the CBD also increases the polarity of the nearest headgroup region of the PCD and the accessibility of small, water-soluble molecules to that region. These findings are in agreement with both calorimetric [25
] and X-ray diffraction data [21
] on Chol/POPS mixtures, which show that the enthalpy of melting and interbilayer spacing continue to change after formation of the CBD (and cholesterol crystals) begins to take place (see also explanation in the end of the paper). Our results and the cited literature [2
] provide proof that the CBD is formed within the PCD as a coexisting domain and continues to exert influence on PCD structure and dynamics. Our results also demonstrate that the CBD, with the lipid-bilayer-like structure, is formed only in the presence of phospholipids in membranes made from mixtures with a Chol/POPS ratio up to ~2.
When the cholesterol content significantly exceeds a Chol/POPS ratio of ~2, cholesterol can be further suspended in buffer. However, the EPR signal of CSL from this suspension is significantly different than that from suspension formed for the Chol/POPS ratio below 2. This signal contains a broad, single-line component, which is identical with the signal obtained for the suspension of pure cholesterol. We conclude that the three-line EPR signals from CSL (as shown in ) characterize the fraction of cholesterol in the lipid-bilayer-like structures that is supported by POPS (in PCD and CBD), while broad, single-line signals (as shown in ) characterize the fraction of cholesterol in solid-state aggregates, presumably outside the PCD and CBD. This broad, single-line signal is completely masked by the strong three-line signal at the Chol/POPS ratio close to 2 (see Sect. 3.4).
The only explanation of the appearance of the broad, single-line signal of CSL in solid-state aggregates of cholesterol is strong spin-spin interaction between CSL molecules. It is likely that during chloroform evaporation, due to differences in polarity and, thus, differences in cholesterol and CSL solubility in chloroform, the local concentration of CSL significantly increases as compared with the concentration of cholesterol. Cholesterol—because it is more polar—crystallizes first, leaving locally concentrated solutions of CSL that enhance spin-spin interaction and broaden the EPR signal. This broadening is not observed in the fraction of cholesterol that forms the CBD supported by the POPS. The observed broad, single-line signal of CSL suggests a different organization of cholesterol in these fractions, with a more rigid structure of solid-state aggregates. It is likely that cholesterol solid-state aggregates have the same structure as cholesterol crystals, as indicated in the literature [20
]. Indeed, DSC measurements (Sect. 3.4) have shown characteristics of the presence of cholesterol crystal transitions in the suspension of cholesterol solid-state aggregates. We have shown that the EPR spin-labeling approach can discriminate the fraction of cholesterol that forms the CBD within the phospholipid bilayer from the fraction that forms cholesterol structures (cholesterol crystals) presumably outside the bilayer. Cholesterol analogue spin labels are superior probes for the structure and dynamics of the CBD. However, they are poor at detecting the organization of cholesterol molecules in cholesterol crystals.
Models created through molecular dynamics simulations (Dr. Marta Pasenkiewicz-Gierula, personal communication) allowed us to compare the organization and dynamics of cholesterol molecules in CBDs with those in PCDs and cholesterol crystals. Some characteristics of cholesterol molecules in the CBD are similar to those in cholesterol crystals (bilayer thickness, surface per cholesterol molecule), while others are similar to those in the PCD (tilt, molecular order parameter). Results showed that the CBD is a dynamic structure with cholesterol mobility similar to the PCD. This is the major difference compared with cholesterol crystals, where cholesterol molecules are immobile.
The only explanation of our results that will not contradict well-documented data from the literature is that when the cholesterol content in the phospholipid bilayer exceeds the CST, both the CBD and cholesterol crystals form in the membrane suspension (). The former is detected by EPR spin-labeling methods, the latter by DSC as well as the X-ray diffraction and MAS NMR methods. The organization of cholesterol molecules in CBDs is similar to that in PCDs (the order and dynamics are similar as well) and different from the rigid structure of cholesterol crystals—although the bilayer structure of the CBD and the pseudo-bilayer structure of cholesterol crystals indicate certain structural similarities between them. However, DSC clearly detects formation of cholesterol crystals and not CBDs, which are fluid cholesterol bilayers with water molecules that have unlimited access to cholesterol –OH groups. In this situation, dehydration of the monohydrate form of cholesterol to the anhydrous form (which, additionally, can stay in this form for a few hours) is not possible. It is difficult to evaluate fractions of cholesterol that form CBDs and cholesterol crystals. Our results show that in POPS membranes the CBD starts to form when the cholesterol content exceeds the solubility threshold and continues to form only to a Chol/POPS mixing ratio of about 2. Further increase in cholesterol content does not increase the amount of cholesterol in the form of the CBD. It is most probable that when the cholesterol content exceeds the CST, parts of cholesterol molecules also form cholesterol crystals.