Cellular systems that control protein dynamism across the membrane

利用模式生物E.在大肠杆菌中，我们研究了介导蛋白质适当生物合成的细胞系统，特别是那些整合到膜中并跨膜转运的蛋白质。我们分析的目标包括Sec机制，允许蛋白质易位和整合，膜相关的蛋白酶是重要的膜蛋白质质量控制以及包膜应激反应，和Dsb系统，引入二硫键到蛋白质。我们已经确定了晶体结构的SecA易位马达和SecYE易位从细菌，嗜热栖热菌。我们确定了SecA-SecY接触残基，发现SecA和SecY以至少两种不同的方式相互作用。我们提出了一种新的机制，SecA将ATP中储存的能量转化为分泌前体蛋白的运动。我们还进行了SecYEG易位子亚基的最近邻分析，以及易位子参与正确折叠的膜蛋白形成的模式的分析。我们发现了一种新的调节机制，其中SecM的阻滞序列与核糖体的出口通道相互作用，从而调节SecA的翻译和折叠。我们对E.其中FtsH、HtpX及其相关因子起重要作用。我们的生物化学和生物学特性的膜整合蛋白酶已扩展到两种酶，RseP和G1pG，催化膜内蛋白水解。最后，我们揭示了DsbA-DsbB-单醌机制的反应机制和潜在的结构基础，该机制产生蛋白质二硫键以引入客户蛋白质。

Using a model organism E. coli, we have investigated the cellular systems that mediate proper biogenesis of proteins, especially those integrated into and transported across membranes. The targets of our analyses included the Sec machinery that allows protein translocation and integration, membrane-associated proteases that are important in membrane protein quality control as well as envelope stress responses, and the Dsb system that introduces disulfide bonds into proteins. We have determined crystal structures of the SecA translocation motor and the SecYE translocon from a bacterium, Thermus thermophilus. We determined the SecA-SecY contact residues and found that SecA and SecY interact with each other in at least two different modes. We proposed a new mechanism, by which SecA converts the energy stored in ATP into the movement of secretory precursor protein. We also carried out nearest neighbor analyses of SecYEG translocon subunits as well as analyses of the mode of involvement of the translocon in the formation of correctly folded membrane proteins. We found a new regulatory mechanism, in which an arrest sequence of SecM interacts with the exit tunnel of the ribosome, thereby regulating translation and folding of SecA. We have characterized the quality control system in the E. coli plasma membrane, in which FtsH, HtpX and associated factors play important roles. Our biochemical and biological characterization of membrane-integrated proteases has extended into two enzymes, RseP and G1pG, which catalyze intramembrane proteolysis. Finally, we have revealed the reaction mechanism and underlying structural bases of the DsbA-DsbB-uniquinone machinery that generates protein disulfide bonds to be introduced into client proteins.