The emergence of resistance to antibiotics by pathogenic organisms is a fundamental human health concern. The solution to this growing epidemic involves a concerted effort to both uncover processes that are critical to cellular viability, and design molecules selective for discrete components within these processes. The Bam complex facilitates the folding and insertion of -barrel proteins into the outer membrane of Gram-negative bacteria. This is an essential but poorly understood process. We seek to explore an early step of the folding mechanism, substrate recognition, because this will provide important insight into a critical cellular process. Further, an understanding of this process will enable the rational development of inhibitors for use as future antibiotics against Gram-negative bacteria. Accordingly, this research studies a protein-protein interaction that is necessary for substrates to be recognized by Bam in E. coli. We will begin by exploring what features of unfolded outer membrane proteins are critical for recruitment to the machine through the use of unfolded BamA as a substrate. Affinity co- purifications and a fluorescence assay will be the principle tools used for this analysis. To characterize a binding site within an implicated lipoprotein component of the Bam complex we will perform mutational analysis of this protein and assay for binding affinity. To verify the conclusions drawn from our biochemical and biophysical data studying BamA as substrate, we will look to identify an analogous signal sequence necessary for recognition by Bam within the essential outer membrane protein LptD. Once our conclusions are recapitulated within the context of this new substrate, we will explore whether the affinity of a substrate for Bam correlates with the amount of folded -barrel proteins found in the OM. Understanding of the substrate-Bam recognition event will prove valuable as we seek to increase efficiency in the expression of native and non-native outer membrane proteins in E. coli. In turn, this technological achievement will prove very enabling for researchers as we seek to develop new antibiotics for treatment of infection by pathogenic organisms. In accordance to the project outline above, we propose the following Specific Aims: Specific Aim I. To characterize requirements for interaction between unfolded BamA and BamD. Specific Aim II. To assess the generality of the ss motif and determine whether it influences protein levels in the OM.