Calorimetric X-Ray Diffraction and Spectroscopic Studies of the Thermotropic Phase Behavior and Organization of Tetramyristoyl Cardiolipin Membranes

摘要

The thermotropic phase behavior and organization of aqueous dispersions of the quadruple-chained, anionic phospholipid tetramyristoyl diphosphatidylglycerol or tetramyristoyl cardiolipin (TMCL) was studied by differential scanning calorimetry, x-ray diffraction, ³¹P NMR, and Fourier-transform infrared (FTIR) spectroscopy. At physiological pH and ionic strength, our calorimetric studies indicate that fully equilibrated aqueous dispersions of TMCL exhibit two thermotropic phase transitions upon heating. The lower temperature transition is much less cooperative but of relatively high enthalpy and exhibits marked cooling hysteresis, whereas the higher temperature transition is much more cooperative and also exhibits a relatively high enthalpy but with no appreciable cooling hysteresis. Also, the properties of these two-phase transitions are sensitive to the ionic strength of the dispersing buffer. Our spectroscopic and x-ray diffraction data indicate that the lower temperature transition corresponds to a lamellar subgel (Lc′) to gel (Lβ) phase transition and the higher temperature endotherm to a Lβ to lamellar liquid-crystalline (Lα) phase transition. At the Lc′/Lβ phase transition, there is a fivefold increase of the thickness of the interlamellar aqueous space from ∼11 Å to ∼50 Å, and this value decreases slightly at the Lβ/Lα phase transition. The bilayer thickness (i.e., the mean phosphate-phosphate distance across the bilayer) increases from 42.8 Å to 43.5 Å at the Lc′/Lβ phase transition, consistent with the loss of the hydrocarbon chain tilt of ∼12°, and decreases to 37.8 Å at the Lβ/Lα phase transition. The calculated cross-sectional areas of the TMCL molecules are ∼79 Ų and ∼83 Ų in the Lc′ and Lβ phases, respectively, and we estimate a value of ∼100 Ų in the Lα phase. The combination of x-ray and FTIR spectroscopic data indicate that in the Lc′ phase, TMCL molecules possess tilted all-trans hydrocarbon chains packed into an orthorhombic subcell in which the zig-zag planes of the chains are parallel, while in the L_β phase the untilted, all-trans hydrocarbon chains possess rotational mobility and are packed into a hexagonal subcell, as are the conformationally disordered hydrocarbon chains in the L_α phase. Our FTIR spectroscopic results demonstrate that the four carbonyl groups of the TMCL molecule become progressively more hydrated as one proceeds from the L_c′ to the L_β and then to the L_α phase, while the two phosphate moieties of the polar headgroup are comparably well hydrated in all three phases. Our ³¹P-NMR results indicate that although the polar headgroup retains some mobility in the L_c′ phase, its motion is much more restricted in the L_β and especially in the L_α phase than that of other phospholipids. We can explain most of our experimental results on the basis of the relatively small size of the polar headgroup of TMCL relative to other phospholipids and the covalent attachment of the two phosphate moieties to a single glycerol moiety, which results in a partially immobilized polar headgroup that is more exposed to the solvent than in other glycerophospholipids. Finally, we discuss the biological relevance of the unique properties of TMCL to the structure and function of cardiolipin-containing biological membranes.