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

革兰氏阴性杆菌铜绿假单胞菌产生多种脂肪酶，其中LIPA通过II型分泌途径分泌到培养上清液中。我们已经证明，这种脂肪酶需要一个名为LIF(脂肪酶特异性折叠酶)的空间伴侣才能获得具有酶活性的构象。目前，脂肪酶折叠的分子机制和折叠酶对脂肪酶分泌的作用尚不清楚。我们建立了脂肪酶LIPA和折叠酶LIF在异源宿主大肠杆菌和同源宿主铜绿假单胞菌中的表达和纯化方法。此外，我们还开发了一种通过体外重折叠失活脂肪酶来测试折叠酶功能的方法。在这里，我们打算重点关注两个主要话题。(1)将研究折叠酶将其底物--失活的脂肪酶转化为具有酶活性的构象的机制。通过各种计算、生物物理、生物化学和生物学方法，在体外分析脂肪酶和折叠酶在折叠过程中的构象变化。具体地说，涉及脂肪酶和折叠酶稳定性和结合的单分子荧光光谱方法将通过基于过滤相关技术和高精度(HP)-FRET的单分子荧光光谱方法以及通过分子动力学模拟、自由能计算和刚性分析来研究折叠酶与其同源脂肪酶之间的相互作用。在人工膜系统中，还将分析折叠酶的膜结合对其动力学和功能的影响。HpFRET将首次对标记在三个位置的脂肪酶-折叠酶系统进行检测。(2)分析了折叠酶与SEC机制相互作用导致脂肪酶LIPA通过细菌内膜转运的分子机制。利用荧光相关光谱和生物化学方法研究SEC-蛋白质和折叠酶的生物相关相互作用。在这里，我们解决的问题是，SecEYG蛋白是否参与了Lif：LIPA复合体的体内解离，以及LIPA如何在分泌过程中识别XCP-机械。这些雄心勃勃的目标只能通过三个小组的密切合作才能实现：Jaeger小组提供分子生物学和生化方法，Seidel小组实施各种荧光光谱方法，Gohlke小组增加计算模型和模拟数据，从而能够从实验和理论上分析折叠酶介导的脂肪酶激活的复杂机制和动力学。

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.