Research efforts involve the modulatory effects of bilayer lipids on the structural reorganizations of integral membrane proteins. Interests lie primarily in characterizing the sizes and formation properties of fluctuating lipid microdomains within biomembranes, using vibrational infrared and Raman spectroscopies and ultrasonic velocimetry techniques. In particular, the compressibilities of systems composed of various lipid microdomains are correlated with intramolecular protein rearrangements. Various reconstituted multilamellar and single shell vesicle assemblies were generated as model systems to demonstrate the effects arising from the lateral compressibility properties of these quantified lipid microaggregates. To study spectroscopically specific bilayer lipid chain order/disorder properties within the membrane microdomains, appropriate lipid acyl chain deuteration was required to allow the vibrational dynamics of the chain moieties to be monitored. Binary and ternary mixtures of saturated chain phosphatidylcholines were specifically examined. Various spectroscopic splitting patterns of the methylene bending modes allowed a determination of lipid microdomain size in terms of the number of acyl chains constituting a given lipid cluster. The compressibilities of the lipid assemblies are to be determined both isothermally and adiabatically. Adiabatic compressibilities of lipid dispersions are determined by ultrasonic velocimetry in which the thermotropic response to the velocity of sound is measured. In examining binary lipid mixtures, microdomain sizes were found to be functions of the lipid mole fractions constituting the system. Specifically, both the lateral compressibilities of the various systems and the integral membrane protein reorganizations are governed by the effective domain sizes defining the assembly. A variety of light scattering studies were also performed on single shell vesicle systems in efforts to correlate size with bilayer microdomain properties as a function of temperature. Results were also obtained which demonstrated the use of vibrational infrared spectroscopy applied toward characterizing lipid microdomain sizes derived from a model raft system consisting of non-hydroxy galactocerebroside, cholesterol, and deuterium labeled dipalmitoylphosphatidylcholine (DPPC-d62) components. The values resulting from the spectroscopic splitting parameters associated with interacting stearoyl chains of GalCer alone permitted the calculation of an aggregate size of the microdomain of about 33 chains;the splitting parameters measured for the sphingosine chains indicated only 2-3 interacting chains. In a 1:1 molar ratio mixture of GalCer with DPPC-d62, the number of interacting sphingosine chains remained about the same, but the number of interacting stearoyl chains decreased to only about 6. Splitting of the bands associated with methylene chains labeled with deuterium in DPPC-d62 indicated a microdomain size of about 11 chains. A marked change in the deformation modes of GalCer methylene chains occurred when a 0.33 mol fraction of cholesterol (Chol) was added to the 1:1 GalCer DPPC-d62 mixture: the splitting associated with microdomain formation was completely abolished, and a band characteristic of hexagonal lipid chain packing, in contrast to the orthorhombic subcell, appeared. The splittings of the methylene chain CD2 bands of DPPC-d62 remained the same although the remaining presence of the unsplit band indicated the existence of both hexagonal and orthorhombic phases. Assesment of the change in frequency and band width of the methylene stretching modes of GalCer and DPPC-d62 with Chol present suggest an increase in chain mobility of GalCer chains but little effect on DPPC. We are interested in determining the precise structural properties of Chol that are important for its marked effect on GalCer methylene chains. In particular, the effect on band splitting of slight modifications in the structure of Chol utilizing compounds related to Chol have been combined with GalCer and DPPC-d62; infrared spectra were measured at 20, -20 and -120. The following steroids have been examined: 5-cholesten-3-α-ol, cholestane, cholestene, dihydrocholesterol, 5-cholesten-3-one, campesterol and lanosterol. Assessments of the results are currently being made. In further elucidating membrane fusion effects, we note that in the absence of intervening proteins, membrane fusion is a multi-step, complex process in which the two membrane bilayers must be brought into close contact after intervening bound water layers have been displaced and the electrostatic repulsions between opposing membranes have been overcome. Although the mechanism of fusion with its several intermediate steps has been intensively studied, less attention has been paid to the initiation of the process;namely, the manner in which close contact occurs. Our focus has been on the reversible aggregation of small phosphatidylcholine single shell vesicles for which close contact between bilayers leads to adhesion of the vesicles but not to fusion. Laser light scattering measurements demonstrated that dilute (1.26 10−5 M) single shell vesicles of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in water (1,790 230 nm in diameter) undergo no significant change in size at room temperature within 41 days. In marked contrast, when DSPC vesicles of this size are cooled at 15 C for 23 hours, the diameter increases nearly 3-fold to about 5,200 nm. Direct microscope observation of the DSPC vesicles at room temperature show single, unaggregated spheres. After being held overnight at 5 C, visual observation reveals that the spheres aggregate into two and three units without fusion, thus confirming the light scattering measurements. The diameters of the aggregated vesicles decrease with increasing temperature;and after the vesicles undergo the gel to liquid crystal phase transition at 54 C, the diameters are reduced markedly to 850 nm at 63.5 C. When cooled to 33 C, the original diameter (1780 240 nm) is regained. On further cooling, the vesicles increase in diameter again;and, if held overnight at 15 C, the diameters return to the value found for the aggregated vesicles. A second cycle of heating and cooling retraces the observations for the DSPC vesicles. Undergoing the phase transition, thus, appears to have had no effect on the ability of the vesicles to aggregate again when exposed to reduced temperature. Vesicle aggregation has been observed with a 10-fold smaller diameter bilayer system. Vesicles of 1.42 10−5 M dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) of 135 35 nm in diameter measured at 20.5 C were cooled to 4.6 C;within 2 hours the diameters increased 10-fold (to 1,340 660 nm), indicating extensive aggregation, which was reversed with increasing temperature. Even at 10 C, the vesicles decreased to about the original size (210 55 nm) before cooling. Had either the DSPC or DPPC vesicles undergone fusion instead of aggregation, heating would not have reversed their status to produce particles of the same size and distribution that existed prior to cooling. In contrast, cooling vesicles of 1.48 10−5 M dimyristoyl-sn-glycero-3-phosphocholine (DMPC) with a diameter at 14 C of 1,100 340 nm to 5.4 C (diameter 1,250 570 nm) is ineffective and the system show no evidence of aggregation. Although composed of neutral lipids, vesicles of DMPC, DPPC and DSPC exhibit a small net negative surface charge at low ionic strength, with DMPC vesicles having the largest negative charge and DSPC vesicles the smallest. The value of the surface charge is thought to reflect the orientation of the polar head group with respect to the bilayer plane, which varies with temperature, ionic strength and the length of the hydrocarbon chain.
