Signal sequences play a central role in the membrane targeting and translocation of nearly all secreted proteins and many integral membrane proteins in both prokaryotes and eukaryotes. This project has as its goal the elucidation of the conformations and interactions of signal sequences as they participate in the steps that make up the export pathway, namely, recognition of a nascent or newly synthesized secretory protein, targeting of the protein to the appropriate membrane, initiation of interactions with lipids and proteins of the membrane- resident translocation apparatus, and translocation of the polypeptide chain across the membrane. We seek a fundamental physical-chemical understanding of how signal sequences may take part in these steps. In the next project period, we will focus on the roles of signal sequences in early steps in export-recognition and targeting. To this end, we will continue studies using synthetic signal peptides and a battery of biophysical and biochemical methods to elucidate the nature of signal sequence binding by two proteins that serve as the first point of recognition of a protein that is destined for the export pathway, namely the signal recognition particle (SRP), which recognizes secretory proteins in eubacteria and targets them to the endoplasmic reticulum membrane, and SecA, the central player in membrane insertion of the polypeptide chain in bacteria, and to relate the findings to the mechanism of protein export in bacteria and mammals. In our studies, we will use Ffh, the E. coli homologue of the mammalian SRP54, as our SRP model. Fluorescence, circular dichroism, nuclear magnetic resonance, and site-specific mutagenesis will be used to determine the conformation of signal peptides upon binding to Ffh and to SecA, and, correspondingly, the structural basis of the protein's ability to bind signal peptides. Furthermore, we will explore the influence of binding to signal sequences and other ligands on the function of Ffh and SecA. Notably, we will explore the relationship of the 4.5S RNA to the structural stability and functions of Ffh. Since all serum antibodies digestive enzymes, and peptide hormones are secreted, as are many other physiologically important proteins, this research will provide important fundamental insight into several biomedical problems. The bacterial secretory apparatus is also a target for new antibiotics, the design of which will be aided by detailed understanding of the key components.
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