SPECTROSCOPY OF BIOMEMBRANE MONOLAYERS

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The general, long-term goal of the research described in this proposal is to use a combined, synergistic, approach to study organized, two- dimensional biomembrane structures at interfaces. The specific objectives of this research project are to combine the methods of biochemistry, surface chemistry, and vibrational spectroscopy for evaluation of structure/function relationships in physiologically relevant in-vitro models for pulmonary lung surfactant. This combined research approach will use biochemical techniques for the isolation and characterization of the newly-discovered plasmalogen phospholipid component of pulmonary surfactant. Classical Langmuir- Blodgett surface chemistry techniques will be used to study phospholipid monolayer structure in surfactant model complexes and in isolated material from native pulmonary surfactant. These experiments, in addition to monitoring phospholipid order and interactions in a physiologically relevant state, will directly test the "squeezing-out" hypothesis of surfactant spreading, namely that one particular component of surfactant becomes enriched at the surface during successive compression cycles, in order to produce near-zero surface tension. Infrared spectroscopy will be used to determine the molecular conformation of these biomembrane models both on solid supports via attenuated total reflectance methods, and in- situ at the air-water interface by external reflectance methods. We will monitor the Ca2+-dependent phospholipid conformation, protein secondary structure, and orientation of ordered peptide segments in Langmuir- Blodgett lipid-peptide films by infrared spectroscopy. This approach will provide dues as to the molecular nature of the interactions between phospholipids, Ca2+, and surfactant proteins. We will study the structure and spatial distribution of the pulmonary surfactant SP-B and SP-C peptides in bilayer ultrathin films using these vibrational spectroscopic methods in combination with surface chemistry techniques. We will further develop a new method for obtaining enhanced IR spectra of biophysical monolayer films in-situ at the air-water interface, and will particularly apply this method to the study of the interaction of surfactant peptides with phospholipid monolayers. A new vibrational spectroscopy approach will also be used in order to identify the two-dimensional micro-domain segregation in binary mixtures used as ultrathin biomembrane models. The combination of these techniques on in-vitro model systems promises to significantly improve our understanding of the structure and reactivity of native pulmonary surfactant in-vivo.