Center for insoluble protein structures (inSPIN), Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10C, DK-8000 Aarhus C., Denmark.
Biophys Chem. 2010 Nov;152(1-3):80-8. doi: 10.1016/j.bpc.2010.08.003. Epub 2010 Aug 13.
We use a cell-free transcription-translation system to monitor the effect of different lipids on the synthesis and folding of the transmembrane domain of the outer membrane protein OmpA from E. coli under physiological conditions. Folding is consistent with previous observations made in vitro at high pH. Synthesis and folding yields are optimal in phosphocholine lipids, particularly in short chain lipids and small vesicles, while lipid rafts do not promote folding compared to the folding in the absence of lipids. Truncated species are observed during translation in the presence of the periplasmic chaperone Skp, which likely binds to the newly synthesized polypeptide chain during cell-free translation and thus prematurely terminate polypeptide chain synthesis. In contrast, folded and unfolded dimers of OmpA correlate negatively with folding yields. This suggests that dimer formation competes with folding and insertion of monomeric OmpA, though folded dimers slowly appear to convert to folded monomers.
我们使用无细胞转录-翻译系统来监测在生理条件下，不同脂质对大肠杆菌外膜蛋白 OmpA 的跨膜结构域的合成和折叠的影响。折叠与先前在高 pH 值下体外观察到的结果一致。在磷酸胆碱脂质中，特别是在短链脂质和小泡中，合成和折叠产率最佳，而与无脂质相比，脂筏并不促进折叠。在周质伴侣 Skp 的存在下，在翻译过程中观察到截短的物种，这可能在无细胞翻译过程中结合到新合成的多肽链上，从而过早终止多肽链的合成。相比之下，OmpA 的折叠和未折叠二聚体与折叠产率呈负相关。这表明二聚体的形成与单体 OmpA 的折叠和插入竞争，尽管折叠的二聚体似乎缓慢地转化为折叠的单体。
