Spectroscopy of Biomembrane Monolayers

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DESCRIPTION (provided by applicant): Pulmonary surfactant is an integral component of mammalian alveoli that serves to reduce surface tension and promote oxygen uptake in the lung. The lack of surfactant leads to respiratory distress syndrome in both infants and adults. In-vivo, surfactant contains four apoproteins, two of which (SP-B and SP-C) have been implicated in the surface-active properties of this material. SP-B has been shown to enhance adsorption, while the alpha-helix form of the SP-C lipoprotein has been shown to maximize hydrophobic interactions with lipids in order to increase the mechanical stability of the monolayer, and catalyze the formation of 3-D lipid-protein reservoirs at the air-water interface. Also, recent research has demonstrated that the deacylated form of SP-C is metastable and undergoes an alpha-helix to beta-sheet rearrangement resulting in protein aggregates that visually resemble amyloid fibrils. These fibrils are found in broncho-alveolar fluid isolated from patients with pulmonary alveolar proteinosis, and consist largely of deacylated SP-C. Progress in isolating specific lipid and protein components of surfactant has advanced to the point where biophysical approaches may be utilized to study specific structure-function relationships. We have applied IR spectroscopy as well as optical microscopy to the study of surfactant model systems in 2-D films. Our results have demonstrated direct evidence for the formation of surface-associated 3-D particles by hydrophobic surfactant proteins. Using a new computational approach, we have identified previously unknown structural intermediates in SP-B and SP-C and followed their reorientation. Furthermore, we have identified pH as one of the mechanisms that influence the formation of amyloid fibrils by deacylated SP-C. In the next grant period, we plan to expand these types of measurements to biophysical monolayers that mimic physiologically relevant pulmonary surfactant models. Using a combination of mutagenesis, vibrational spectroscopy and optical microscopy, we will examine the particular structural mechanisms by which SP-B influences monolayer surface activity as well as the mechanisms by which deacylated SP-C forms amyloid fibrils.