The mechanism by which protein molecules are localized in various compartments and membranes of the cell has stood as one of the great problems of cell biology. It is now clear that nascent polypeptide chains are carried into the lumen of that organelle where the leader (signal) sequence is removed. Completed polypeptides travel to the Golgi body where they are targeted to specific organelles. Plasma membrane receptors such as the low density lipoprotein (LDL) receptor are cycled from the plasma membrane through coated endocytic vesicles and endosomes, delivering LDL particles to the lysosomes while the receptors return to the cell surface. In humans, errors in the targeting, processing and recycling of LDL receptors lead to familial hypercholesterolemia resulting in premature atherosclerosis. The general goals of this research program are to probe the physical and chemical basis by which membrane proteins attain and maintain defined three-demensional structures in biological membranes, the mechanism by which such proteins carry out their biological function and the role of electric fields in these processes. The best starting point for such investigations is a detailed three-dimensional structure derived from diffraction studies at a resolution consistent with atomic modeling.
The specific aims of this research program are directed at two aspects of these general problems. First, the closely related membrane active peptides: alamethicin and suzukacillin will be studied as examples of proteins which can spontaneously integrate into lipid bilayers from aqueous solution. We plan to characterize the structure of these peptides in crystals grown from aqueous solution to define the molecular surface which interacts with the lipid bilayer. EM image reconstruction techniques will be applied to two dimensional arrays of gated and ungated peptide channels to define the structural basis of the voltage-gating process in these systems. Secondly, we plan to prepare three-dmensional crystals of E. coli leader peptidase, and to characterize its molecular structure at high resolution in an effort to define the structural basis of leader peptide recognition and cleavage, and the role of this process in membrane protein biogenesis.
