Function of lipase-specific foldase for folding and secretion of a lipase from Pseudomonas aeruginosa

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The Gram-negative bacterium Pseudomonas aeruginosa produces several lipases, among them LipA which is secreted into the culture supernatant via the type II secretion pathway. We have demonstrated that this lipase requires a steric chaperone named Lif (lipase-specific foldase) to achieve an enzymatically active conformation. At present, the molecular mechanism of lipase folding and the role of the foldase for lipase secretion are unknown. We have established methods for expression and purification of the lipase LipA and the foldase Lif in the heterologous host Escherichia coli and in the homologous host P. aeruginosa. Furthermore, we have developed a protocol allowing to test foldase functionality by in vitro refolding of inactive lipase. Here, we intend to focus on two major topics. (1) The mechanisms by which the foldase converts its substrate, inactive lipase, into an enzymatically active conformation will be studied. The conformational changes of lipase and foldase along the folding pathway will be analyzed in vitro by various computational, biophysical, biochemical and biological methods. Specifically, interactions between the foldase and its cognate lipase related to the stability and binding of lipase and foldase will be studied by single-molecule fluorescence spectroscopic methods based on filtered correlation techniques and high precision (hp)-FRET and by molecular dynamics simulations, free energy calculations, and rigidity analyses. The influence of the membrane association of the foldase on its dynamics and function will also be analyzed in artificial membrane systems. For the first time, the lipase-foldase system labeled at three positions with fluorescence markers will be examined by hpFRET. (2) The molecular mechanism of interaction between the foldase and the Sec-machinery resulting in translocation of the lipase LipA through the bacterial inner membrane will be analyzed. Biological relevant interactions of Sec-proteins and foldase will be studied by fluorescence correlation spectroscopy and biochemical methods. Here, we address the questions if SecEYG proteins are involved in the in vivo dissociation of the Lif:LipA complex and how LipA recognizes the Xcp-machinery during the secretion. These ambitious goals can only be reached by a close collaboration between three groups: the Jaeger group provides molecular biological and biochemical methods, the Seidel group carries out various fluorescence spectroscopic methods and the Gohlke group adds computational modeling and simulation data thereby allowing to experimentally and theoretically analyze the complex mechanism and dynamics of foldase-mediated lipase activation.