Time-resolved distance determination by tryptophan fluorescence quenching: Probing intermediates in membrane protein folding
Time-resolved distance determination by tryptophan fluorescence quenching: Probing intermediates in membrane protein folding
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DOI：
10.1021/bi9824644
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发表时间：
1999-04-20
期刊：
影响因子：
2.9
通讯作者：
Tamm, LK
中科院分区：
文献类型：
--
作者：
Kleinschmidt, JH;Tamm, LK
利用大肠杆菌外膜蛋白A（OmpA）研究了整合膜蛋白的插入和折叠机制。这项工作描述了一种新的方法来解决这个问题，通过结合结构信息从色氨酸荧光淬灭在不同深度的脂质双层与动力学的重折叠过程。在2和40摄氏度之间的温度范围内进行的实验使我们能够检测，捕获和表征以前未识别的折叠中间体的OmpA插入和折叠成脂质双层的途径。三个膜结合的中间体被发现，其中的Trps的平均距离分别为14-16，10-11，和0-5埃，从双层中心。第一折叠中间体在2 ℃下稳定至少1小时。第二中间体已在7至20 ℃的温度下分离。在此阶段，Trps向双层中心移动4-5埃。随后，在26-28 ℃下可观察到的中间体中，Trps向双层中心再移动5-10埃。在较高的重折叠温度下观察到最终（天然）结构。在这种结构中，Trps位于离双层中心平均约9-10埃的位置。监测的演变色氨酸荧光淬灭的溴化脂质在不同温度下的重折叠过程中，因此，使我们能够识别和表征中间状态的折叠过程中的一个完整的膜蛋白。
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 Angstrom, 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 Angstrom 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 Angstrom 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 Angstrom 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.