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 spectroscopies and 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. Adiabatic compressibilities of 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. 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 a membranes hydrophobic core. In the presence of Mg(2+) DPPS maintains an ordered orthorhombic subcell gel phase though the phase transition temperature, while the DPPC component is only minimally perturbed with respect to the gel to liquid crystalline phase change. The DPPC matrix exhibits no response other than to rearrange its acyl chains in a manner consistent with the DPPS matrix. The addition of Mg(2+) induces a reorganization of the lipid domains in which the gel phase acy chain planes rearrange from an hexagonal configuration toward a triclinic, parallel chain subcell. Examination of the acyl chain methylene deformation modes at low temperatures allows a determination of DPPS microdomain sizes, which decrease upon the addition of DPPC in the absence of the magnesium ion. On adding Mg(2+), a uniform DPPS size is observed in the binary mixtures. In this system, microdomains are distributed throughout the bilayer. Light scattering and fluorescence measurements indicated that magnesium induces both the aggregation and the fusion of the lipid assemblies as a function of the ratio of DPPS to DPPC. In summary, the lipid reorganizations within predominantly DPPS microdomains represent a mechanistically important aspect for disrupting the critical fusion intermediate, the hemifusion diaphragm, in route to complete membrane fusion.

Project Start
Project End
Budget Start
Budget End
Support Year
37
Fiscal Year
2009
Total Cost
$1,316,568
Indirect Cost
City
State
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Howard, Frank B; Levin, Ira W (2010) Lipid vesicle aggregation induced by cooling. Int J Mol Sci 11:754-61
Schultz, Zachary D; Pazos, Ileana M; McNeil-Watson, Fraser K et al. (2009) Magnesium-induced lipid bilayer microdomain reorganizations: implications for membrane fusion. J Phys Chem B 113:9932-41
Crane, Nicole J; McHone, Ben; Hawksworth, Jason et al. (2008) Enhanced surgical imaging: laparoscopic vessel identification and assessment of tissue oxygenation. J Am Coll Surg 206:1159-66
Schultz, Zachary D; Stranick, Stephan J; Levin, Ira W (2008) Tip-enhanced Raman spectroscopy and imaging: an apical illumination geometry. Appl Spectrosc 62:1173-9
Schultz, Zachary D; Levin, Ira W (2008) Lipid microdomain formation: characterization by infrared spectroscopy and ultrasonic velocimetry. Biophys J 94:3104-14
Chen, Tsoching; Lee, Min-Jung; Kim, Yeong Sang et al. (2008) Pharmacodynamic assessment of histone deacetylase inhibitors: infrared vibrational spectroscopic imaging of protein acetylation. Anal Chem 80:6390-6