Marti T
Department of Molecular Biology, Bernhard Nocht Institute, Bernhard-Nocht-Strasse 74, D-20359 Hamburg, Germany.
J Biol Chem. 1998 Apr 10;273(15):9312-22. doi: 10.1074/jbc.273.15.9312.
Bacteriorhodopsin is a heptahelical membrane protein that can be refolded to the native state following denaturation. To analyze the in vitro folding process with independent structural domains, eight fragments comprising two (AB, FG), three (AC, EG), four (AD, DG) or five (AE, CG) of the transmembrane segments were produced by expression in Escherichia coli. The polypeptides were purified to homogeneity by solvent extraction of E. coli membranes, repeated phase separation, and anion-exchange chromatography employing the C-terminal tail of bacteriorhodopsin for adsorption. Upon reconstitution into phospholipid/detergent micelles pairs of complementary fragments (AB.CG, AC.DG, AD.EG, and AE.FG) assembled in the presence of retinal to regenerate the characteristic bacteriorhodopsin chromophore with high efficiency. Together with previous studies, these results demonstrate that the covalent connections in each of the six interhelical loops are dispensable for a correct association of the helices. The different loops, however, contribute to the stability of the folded structure, as shown by increased susceptibilities toward denaturation in SDS and at acidic pH, and decreased Schiff base pKa values for the AB.CG, AC. DG, AD.EG, and AE.FG complexes, compared with the intact protein. Notably, the heptahelical bundle structure was also generated by all possible combinations of pairs of overlapping fragments, containing one (AC.CG, AD.DG, AE.EG), two (AD.CG, AE.DG), or three (AE.CG) redundant helices. The spectral properties of the chromophores indicate that the retinal-binding pocket of the AC.CG, AD.CG, and AE. CG complexes is formed by helices A and B of the respective N-terminal fragment and the C-terminal CG fragment, whereas the AD. DG, AE.DG, and AE.EG complexes are likely to adopt a heptahelical bundle structure analogous to AD.EG. The combined data show that the specificity of the helix assembly of bacteriorhodopsin is influenced by connectivities provided by the C-D and E-F surface loops.
细菌视紫红质是一种七螺旋膜蛋白，变性后可重新折叠成天然状态。为了用独立的结构域分析体外折叠过程，通过在大肠杆菌中表达产生了包含两个（AB、FG）、三个（AC、EG）、四个（AD、DG）或五个（AE、CG）跨膜片段的八个片段。通过溶剂萃取大肠杆菌膜、重复相分离以及使用细菌视紫红质的C末端尾巴进行吸附的阴离子交换色谱法，将这些多肽纯化至同质。在视网膜存在的情况下，互补片段（AB.CG、AC.DG、AD.EG和AE.FG）对重构到磷脂/去污剂胶束中，能够高效地组装并再生出具有特征性的细菌视紫红质发色团。与先前的研究一起，这些结果表明，六个螺旋间环中每个环的共价连接对于螺旋的正确缔合是可有可无的。然而，不同的环有助于折叠结构的稳定性，这表现为在SDS中以及在酸性pH下对变性的敏感性增加，并且与完整蛋白质相比，AB.CG、AC.DG、AD.EG和AE.FG复合物的席夫碱pKa值降低。值得注意的是，七螺旋束结构也由包含一个（AC.CG、AD.DG、AE.EG）、两个（AD.CG、AE.DG）或三个（AE.CG）冗余螺旋的重叠片段对的所有可能组合产生。发色团的光谱特性表明，AC.CG、AD.CG和AE.CG复合物的视黄醛结合口袋由相应N末端片段的A和B螺旋以及C末端CG片段形成，而AD.DG、AE.DG和AE.EG复合物可能采用类似于AD.EG的七螺旋束结构。综合数据表明，细菌视紫红质螺旋组装的特异性受C-D和E-F表面环提供的连接性影响。
