Calorimetric, x-ray diffraction, and spectroscopic studies of the thermotropic phase behavior and organization of tetramyristoyl cardiolipin membranes

Abstract

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, (31)P 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 (L(c)') to gel (L(beta)) phase transition and the higher temperature endotherm to a L(beta) to lamellar liquid-crystalline (L(alpha)) phase transition. At the L(c)'/L(beta) phase transition, there is a fivefold increase of the thickness of the interlamellar aqueous space from approximately 11 A to approximately 50 A, and this value decreases slightly at the L(beta)/L(alpha) phase transition. The bilayer thickness (i.e., the mean phosphate-phosphate distance across the bilayer) increases from 42.8 A to 43.5 A at the L(c)'/L(beta) phase transition, consistent with the loss of the hydrocarbon chain tilt of approximately 12 degrees , and decreases to 37.8 A at the L(beta)/L(alpha) phase transition. The calculated cross-sectional areas of the TMCL molecules are approximately 79 A(2) and approximately 83 A(2) in the L(c)' and L(beta) phases, respectively, and we estimate a value of approximately 100 A(2) in the L(alpha) phase. The combination of x-ray and FTIR spectroscopic data indicate that in the L(c)' 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(beta) 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(alpha) 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(beta) and then to the L(alpha) phase, while the two phosphate moieties of the polar headgroup are comparably well hydrated in all three phases. Our (31)P-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(beta) and especially in the L(alpha) 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.

FIGURE 1

DSC thermograms illustrating the polymorphic phase behavior exhibited by dispersions of TMCL in Tris buffer (see Materials and Methods). The heating scan A was obtained after low-temperature equilibration of the sample as described in Materials and Methods. Cooling scans B and D were obtained upon cooling from high temperature to temperatures near −7°C and 18°C, respectively. Heating scan C was obtained by immediately reheating the sample after cooling to temperatures near −7°C, whereas heating scan E was obtained by immediate reheating the sample after cooling to temperatures near 18°C. These data were acquired with the Multi-Cell DSC at a scanning rate of 10°C h⁻¹.

FIGURE 2

DSC thermograms illustrating the polymorphic phase behavior exhibited by dispersions of TMCL in sodium phosphate buffer (see Materials and Methods). Heating scan A represents the pattern of behavior observed upon initial heating of samples that were previously extensively incubated at low temperatures. The other thermograms are typical of those obtained upon subsequent cooling (B) and reheating (C) between 0°C and 55°C. These data were acquired with the Microcal DSC at a scanning rate of 60°C h⁻¹.

FIGURE 3

Scattered x-ray intensity of TMCL in small-angle (A) and wide-angle regime (B) at three temperatures in the L_c′ (2°C), L_β (35°C), and L_α (55°C) phase. Data shown were acquired during heating using samples that were initially fully equilibrated at low temperatures. Arrows in panel A indicate the position of the first-order Bragg peak except for the pattern at 2°C, which has an additional arrow marking the fourth diffraction order. Solid lines show the best fits to the SAXS data applying a global analysis technique (see Materials and Methods). Bracketed numbers in panel B give the Miller indices of the corresponding Bragg rods.

FIGURE 4

Low-resolution, one-dimensional electron density profiles along the bilayer normal calculated from the small-angle scattering diffraction patterns exhibited by aqueous dispersions of TMCL. Electron density profiles were calculated for (A) L_c′ phase at 2°C; (B) L_β phase at 35°C; and (C) L_α phase at 55°C.

FIGURE 5

FTIR spectra observed upon heating fully equilibrated aqueous dispersions of TMCL. The absorbance spectra shown were acquired at the temperatures indicated and typify the C-H stretching (A), the C=O stretching (B), the CH₂ scissoring (C), and the asymmetric phosphate stretching (D) regions of the IR spectra of this compound at temperatures bracketing the two calorimetrically resolved thermotropic phase transitions. The vertical dashed line (A) marks the position of the maxima of CH₂ symmetric stretching band observed in the in the three polymorphic forms of this lipid. The dashed lines (B) represent the subcomponents of the observed absorption band. The asterisks (D) mark the positions of the peaks of the CH₂ wagging band progression.

FIGURE 6

Proton-decoupled ³¹P-NMR powder patterns exhibited by aqueous dispersions of TMCL. The spectra shown were acquired in the heating mode after prolonged sample equilibration at low temperatures and are representative of (A) L_c′ phase at 2°C; (B) L_β phase at 32°C; and (C) L_α phase at 48°C.