LIPID CONTROL OF MEMBRANE PROTEIN ORGANIZATION

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The long term objective of this work is to determine how the structures of lipids and proteins are altered upon their mutual interaction, and how these alterations are related to the biochemical functions of membrane proteins, and to the physiological functions of lung surfactant. Principles extracted from specific examples strengthen the basis for understanding the organization of biological membranes and how this organization may be altered during pathological states. Three specific aims will be pursued: (1) To determine precise acyl chain rotamer population in biologically relevant, conformationally disordered phospholipid phases. To achieve Aim 1, two new Fourier-transform infrared (FT-IR) methods will be applied for the quantitative, position-dependent determination of acyl chain trans- gauche isomerization and for the evaluation of specific disordered forms (kinks, double gauche, etc.). (2) To determine how the presence of major membrane components alters the distribution of conformational states available to phospholipids. To achieve Aim 2, the FT-IR approaches will be applied to reconstituted binary systems of increasing complexity (phospholipid/cholesterol or phospholipid/CaATPase) and finally to an intact organism (A. laidlawii) where the chain lengths in the plasma membrane can be controlled. (3) To use the structural insights gained in Aims 1 and 2 for evaluation of structure/function relationships in a physiologically essential, yet biophysically still manageable system, lung surfactant, to be studied in vitro. The major surfactant proteins will be isolated and reconstituted into appropriate lipid mixtures. Protein secondary structure and orientation of the ordered segments in thin lipid films will be monitored with polarized attenuated total reflectance FT-IR. Phospholipid monolayer structure in native surfactant and in reconstituted systems will be monitored with the novel technique of FT-IR external reflection spectroscopy in situ at the air-water interface. The experiments will directly test the "squeezing-out" hypothesis of surfactant function, namely that DPPC, the main lipid component, becomes enriched at the surface during successive compression cycles, in order to produce the requisite zero surface tension.