This is part of a collaborative project between a bacterial geneticist (Dr. Silhavy) and a physical chemist (Dr. Gierasch) to study the molecular functions of the signal sequence in protein export. Most exported proteins are synthesized initially in precursor form with an amino-terminal signal sequence. Convincing evidence demonstrating the essential role of this sequence in the export process has been presented. However, details of its function(s) are not well understood. Because of its small size (15-30 amino acids), this export signal is particularly amenable to analysis using modern genetic and physical techniques, such as gene fusions that specify hybrid proteins, site-specific mutagenesis using synthetic oligonucleotides, solid-state peptide synthesis, and fluorescence enregy transfer and quenching in model membrane systems. Previous results from the laboratories of these researchers demonstrated that intrinsic physical-chemical properties of the signal sequence are correlated with its in vivo function. This project will expand and amplify the ongoing interdisciplinary approach to dissect the molecular mechanism by which the signal sequence of exported proteins interacts with a biological membrane. This goal will be facilitated through the use of export-defective mutant strains of E. coli in which the signal sequence of the outer membrane protein, LamB, is altered, and through the physical-chemical study of corresponding synthetic signal peptides.
