Structural Requirements for Protein Membrane Assembly

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9316891 Dalbey The proposed research is focused on how N-terminal tails of bacterial membrane proteins are transferred across the membrane bilayer. It is not known whether membrane insertion of these tails will require SecA or SecY, central components of the sec- machinery, or whether insertion can occur spontaneously into the lipid bilayer. Most proteins with N-terminal periplasmic tails conform to the "positive inside" rule with mroe positively charged residues on cytosolic loops compared to periplasmic loops. However, subunit H of the photosynthetic reaction center of Rhodobacter sphaeroides, which contains an N-terminal periplasmic tail, violates this rule and has a highly negatively charged region at the membrane/cytoplasm border. The aims of this proposal are: 1), to determine whether short N-terminal tails are inserted through the lipid or through a proteinaceous complex within the membrane; 2), to define the features (length, charge, or an amphiphilic helix) within N-terminal tails that are needed to translocate the tails across the membrane; 3), to examine whether there is a certain length of the N-terminal tails where the membrane proteins utilize the sec- machinery; and 4), to identify the determinants of the protein topology of subunit H, a protein that has an orientation that disobeys the "positive inside" rule. A variety of approaches will be used, including site-directed mutagenesis, gene fusions, topological mapping, and biochemical studies with protein- free liposomes. %%% This project focuses on the question of how bacterial membrane proteins are inserted into membranes. The delicate architecture of biological membranes, and the fine- resolution accuracy of placement of membrane proteins in biological membranes, are absolutely critical to the proper functioning of biological systems. An understanding of the "rules" that Nature uses to achieve this defined architecture is in turn critical to the successful exploitation of modern gen etic engineering and bioprocessing technologies. This research projects will result in better understanding those rules of Nature concerning how membrane proteins achieve their final topology and location. ***