Kleinschmidt J H, Tamm L K
Department of Molecular Physiology and Biological Physics, Center for Structural Biology, University of Virginia Health Sciences Center, Charlottesville 22906-0011, USA.
Biochemistry. 1999 Apr 20;38(16):4996-5005. doi: 10.1021/bi9824644.
The mechanism of insertion and folding of an integral membrane protein has been investigated with the beta-barrel forming outer membrane protein A (OmpA) of Escherichia coli. This work describes a new approach to this problem by combining structural information obtained from tryptophan fluorescence quenching at different depths in the lipid bilayer with the kinetics of the refolding process. Experiments carried out over a temperature range between 2 and 40 degrees C allowed us to detect, trap, and characterize previously unidentified folding intermediates on the pathway of OmpA insertion and folding into lipid bilayers. Three membrane-bound intermediates were found in which the average distances of the Trps were 14-16, 10-11, and 0-5 A, respectively, from the bilayer center. The first folding intermediate is stable at 2 degrees C for at least 1 h. A second intermediate has been isolated at temperatures between 7 and 20 degrees C. The Trps move 4-5 A closer to the center of the bilayer at this stage. Subsequently, in an intermediate that is observable at 26-28 degrees C, the Trps move another 5-10 A closer to the center of the bilayer. The final (native) structure is observed at higher temperatures of refolding. In this structure, the Trps are located on average about 9-10 A from the bilayer center. Monitoring the evolution of Trp fluorescence quenching by a set of brominated lipids during refolding at various temperatures therefore allowed us to identify and characterize intermediate states in the folding process of an integral membrane protein.
利用大肠杆菌的β桶形成外膜蛋白A（OmpA）对整合膜蛋白的插入和折叠机制进行了研究。这项工作描述了一种解决该问题的新方法，即将从脂质双层不同深度处的色氨酸荧光猝灭获得的结构信息与重折叠过程的动力学相结合。在2至40摄氏度的温度范围内进行的实验使我们能够检测、捕获并表征OmpA插入脂质双层并折叠的途径上先前未鉴定的折叠中间体。发现了三种膜结合中间体，其中色氨酸与双层中心的平均距离分别为14 - 16埃、10 - 11埃和0 - 5埃。第一个折叠中间体在2摄氏度下稳定至少1小时。在7至20摄氏度之间的温度下分离出了第二个中间体。在此阶段，色氨酸向双层中心移动4 - 5埃。随后，在26 - 28摄氏度下可观察到的一个中间体中，色氨酸又向双层中心移动5 - 10埃。在更高的重折叠温度下观察到最终（天然）结构。在这种结构中，色氨酸与双层中心的平均距离约为9 - 10埃。因此，通过在不同温度下重折叠过程中一组溴化脂质监测色氨酸荧光猝灭的演变，使我们能够识别和表征整合膜蛋白折叠过程中的中间状态。
