CAA: Synthetic Signal Peptides: Probes of Molecular Interactions During Secretion

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9629740 Kendall This is a Career Advancement Award for Women Scientists and Engineers. This project concerns how proteins are targeted to and transported across cell membranes. In most cases, whether the membrane in question is the bacterial membrane, the eukaryotic endoplasmic reticulum, or the mitochondrial or chloroplast membrane, a unifying feature of such protein transport systems is the involvement of an amino-terminus signal peptide on the protein to be transported. In the case of transport across the eukaryotic endoplasmic reticulum, the signal peptide interacts with a signal recognition particle (SRP) prior to the initiation of transport. The proposed studies are aimed at elucidating the interaction between the signal peptide of a bacterial secreted protein and P48 (also called Ffh), the Escherischia coli homologue of the 54 kD protein of the mammalian signal recognition particle (SRP). Like its mammalian counterpart, there is growing evidence that P48 plays a vital role in the initial stages of protein secretion through recognition of the signal peptide region of export-competent proteins. This interaction will be investigated using a series of model synthetic signal peptides which vary systematically in their degree of hydrophobicity and which represent different levels of secretion competence in vivo. These peptides will be chemically synthesized, purified, and characterized to verify their physical properties, including secondary structure. A binding assay will be developed based on displacement of a radiolabeled peptide. A Scatchard analysis will be used to determine the dissociation constant for the signal peptide-P48 pair under different conditions, including the association of 4.5S RNA and the present of various forms of guanine nucleotides. Subsequently,, a chemical crosslinking approach will be used to map the location on P48 involved in the signal peptide interaction. These studies will help in our understanding of early events in protein secretion in E. coli and in the several other systems which have been found to have an SRP homologue. ***