of Work: Our research efforts involve the modulatory effects of bilayer lipids on the structural reorganizations of integral membrane proteins. Our interest lay primarily in characterizing the sizes and formation properties of fluctuating lipid microdomains within biomembranes, using vibrational infrared and Raman spectroscopy ultrasonic velocimetry techniques. In particular, the compressibilities of systems composed of various lipid microdomains were 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 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 were determined both isothermally and adiabatically. An infrared diamond anvil cell was used to measure bilayer isothermal compressibility. Pressures were defined by monitoring the spectra of a pressure transducing material, while volume changes were measured directly. Adiabatic compressibilities of the lipid dispersions were 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, the lateral compressibilities of the binary systems and integral membrane protein reorganizations were 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 dipalmitoylphosphatidylcholine components. The resulting spectroscopic correlation field components of the lipid acyl chain CH2 methylene deformation modes, observed when lipid multilamellar assemblies are rapidly frozen from the liquid crystalline state to the gel phase, indicated the existence of lipid microdomains at the several nanometer scale. The addition of cholesterol disrupts the glycosphingolipid selectively, in contrast to perturbing the disaturated chain phospholipid matrix. The sizes of the aggregates were determned from the correlation field effects of interacting acyl chains at low temperatures. Complementary acoustic velocimetry measurements indicated that the microdomain formation decreases the total volume adiabatic compressibilities of the multilamellar vesicle assemblies. Addition of cholesterol, however, disrupts the galactocerebroside domains, resulting in a slight increase in the lipid assemblies total adiabatic compressibility. The combination of these two physical approaches offers new insights into microdomain formation and their properties in model bilayer systems. For understanding more completely the steps involved in the transition of two contiguous bilayers as they fuse under the influence of a fusogenic agent, such as the magnesium ion, we emphasize the use of infrared spectroscopic techniques for a detailed characterization of lipid bilayer fusion properties. In particular, we examined the binary DPPS/DPPC bilayer system both to assess lipid microdomain formation and acyl chain rearrangements within the membranes hydrophobic core. In this system, microdomains are distributed throughout the bilayer. In the presence of the magnesium cation , DPPS maintains a gel phase configuration above the phase transition, while DPPC exhibit no response other than to rearrange their acyl chains in a manner consistent with the DPPS matrix. In summary, the lipid reorganizations within predominantly DPPS microdomains represent a critical aspect for disrupting the critical fusion intermediate, the hemifusion diaphragm, in route to complete membrane fusion.
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