A10: Membrane translocation and chaperone-dependent folding of the lipases A of Pseudomonas aeruginosa
Protein secretion in bacteria follows a multitude of pathways facilitated by machineries of different complexities for translocation of unfolded and folded proteins. In Gram-negative bacteria the outer membrane and the intermembrane periplasm form barriers for secretion, but also provide unique environments for protein maturation and folding. Bacterial lipases, such as lipase A of Pseudomonas aeruginosa (LipA), represent an important and a ubiquitous class of secreted proteins, which have an outstanding biotechnological and, as virulence factors, a biomedical importance. Before being extracellularly secreted via the type II secretion system (T2SS), the precursor form of LipA is translocated into the periplasm by the Sec machinery and then folded into a functional form. The crucial folding step is assisted by the dedicated foldase chaperone LipH. Recently, an effect of LipH on Sec-driven translocation of LipA has been revealed, suggesting a cooperation of these two machineries, possibly in a form of a functional membrane-embedded complex. Here, we propose to combine biochemical, biophysical and structural biology approaches to investigate the mechanism of Sec-mediated LipA translocation and to examine the role of LipH in transport and folding of LipA at the membrane interface in vitro using a membrane-reconstituted secretion system of Pseudomonas. Furthermore, we set out to investigate the effect of the native-like interfacial membrane crowding on LipH:LipA recognition, folding and dissociation reactions by using custom-tailored membranes decorated by native and synthetic crowding agents. The results will provide us with a view on the targeting, membrane translocation and folding of LipA under near-native conditions, which will be integrated in a comprehensive model of the Sec/T2SS secretion pathway indispensable for the virulence of every bacterial pathogen.
Project leader: Jun.-Prof. Dr. Alexej Kedrov, , Group "Synthetic Membrane Systems"
Researcher: Athanasios Papadopoulos,