This proposal addresses a general objective of central importance in current biophysics-namely to determine those structural and dynamic features of phospholipid molecules that govern the distribution and function of membrane-bound proteins. This objective will be approached through three specific aims: 1) To determine whether membrane proteins partition into regions of particular chemical structure or physical order in complex lipid environments, and to determine how the above partitioning characteristics affect the function of membrane-bound enzymes. 2) To determine the magnitude and nature of membrane protein-induced perturbation at specific sites on phospholipid molecules. 3) To determine the conformational changes that occur in both the lipid and protein components upon their mutual interaction in model vesicle systems and in a reasonably simple native system (pulmonary surfactant) to be studied in vitro. The proteins to be used are glycophorin, a well-characterized structural protein from the erythrocyte membrane, and CaATPase from rabbit sarcoplasmic reticulum, a membrane-bound enzyme with several well-defined functions. The physical methods to be used to address the specific aims are Raman and Fourier Transform Infrared spectroscopies and Differential Scanning Calorimetry. Purified proteins will be reconstituted into binary lipid mixtures selected to mimic the phase properties of natural membranes and to be amenable to spectroscopic analysis. Parallel enzyme activity and physical measurement will be used to address Aim 1. Structural studies of proteins reconstituted with phospholipids modified at particular sites will be used to address Aim 2. The components crucial to the function of pulmonary surfactant will be studied individually and in combination using attenuated total reflectance IR spectroscopy in order to understand structure/function relationships (Aim 3).
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