Intellectual Merit: The envelope of Gram-negative bacteria consists of two membranes separated by the periplasmic compartment that contains the peptidoglycan wall. The inner membrane is in contact with the cytosol while the outer membrane contacts the extracellular environment. The OM is a unique structure, essential for Gram-negative bacteria, composed of lipopolysaccharide (LPS), phospholipids and proteins. It is a very selective permeability barrier that allows the bacteria to survive in hostile environments. Outer membrane proteins (OMPs) are integral membrane proteins with beta-barrel structures embedded in the outer membrane. Among their many functions, some OMPs are porins mediating the selective permeability of the membrane while others serve as adhesins responsible for adhesion and colonization of host tissues. OMPs are synthesized in the cytosol and translocated across the inner membrane by the SEC translocation machinery. However, how these hydrophobic proteins cross the periplasm and insert specifically into the OM and fold into their typical b-barrel structure is poorly understood. A number of periplasmic proteins have been implicated in the transport and insertion of OMPs. This project is focused on understanding the role of the "Seventeen Kilodalton Protein" (Skp); an ATP-independent periplasmic chaperone that prevents protein aggregation and is proposed to escort OMPs across the periplasm and assists in their insertion into membranes. A combination of crystallographic, NMR, electron microscopy and biochemical approaches will be used to determine how Skp binds its cargo proteins and prevents their aggregation. NMR techniques will be used to test whether the cargo proteins undergo folding while bound to the chaperone or if they are kept in an unfolded state until delivered to the outer membrane. Since Skp is ATP-independent, insights into the mechanisms of cargo release will be obtained by biochemically probing the hypothesis that LPS binding to Skp triggers cargo release. Mutagenesis followed by in vitro and in vivo assays will then be used to test this hypothesis. The successful completion of these experiments will break new ground in the understanding of how proteins are delivered and folded into the bacterial outer membrane. The study of Skp will provide insights into the general mechanism of ATP-independent chaperones such as Prefoldin. In addition, the lessons learned from the Gram-negative Skp system may provide valuable clues for the mechanism of protein folding and insertion in the mitochondrial outer membrane.
Broader Impacts: The educational program describes several strategies to increase the engagement of underrepresented groups in science including teaching, outreach and mentoring components. In addition the program integrates a plan to foster research-based critical thinking in undergraduate biochemistry classes. The research makes use of multi-user facilities such as synchrotron sources and inter-institutional NMR facilities. This provides outstanding training opportunities in cutting edge technologies for graduate and undergraduate students.
