Hunt J F, Earnest T N, Bousché O, Kalghatgi K, Reilly K, Horváth C, Rothschild K J, Engelman D M
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511, USA.
Biochemistry. 1997 Dec 9;36(49):15156-76. doi: 10.1021/bi970146j.
In order to characterize the thermodynamic constraints on the process of integral membrane protein folding and assembly, we have conducted a biophysical dissection of the structure of bacteriorhodopsin (BR), a prototypical alpha-helical integral membrane protein. Seven polypeptides were synthesized, corresponding to each of the seven transmembrane alpha-helices in BR, and the structure of each individual polypeptide was characterized in reconstituted phospholipid vesicles. Five of the seven polypeptides form stable transmembrane alpha-helices in isolation from the remainder of the tertiary structure of BR. However, using our reconstitution protocols, the polypeptide corresponding to the F helix in BR does not form any stable secondary structure in reconstituted vesicles, and the polypeptide corresponding to the G helix forms a hyperstable beta-sheet structure with its strands oriented perpendicular to the plane of the membrane. [The polypeptide corresponding to the C helix spontaneously equilibrates in a pH-dependent manner between a transmembrane alpha-helical conformation, a peripherally bound nonhelical conformation, and a fully water soluble conformation; the conformational properties of this polypeptide are the subject of the accompanying paper: Hunt et al. (1997) Biochemistry 36, 15177-15192.] Our observations suggest that the folding of alpha-helical integral membrane proteins may proceed spontaneously. However, the preference for a non-native conformation exhibited by two of the polypeptides suggests that the formation of some transmembrane substructures could require external constraints such as the links between the helices, interactions with the rest of the protein, or the involvement of cellular chaperones or translocases. Our results also suggest a strategy for improving the thermodynamic stability of alpha-helical integral membrane proteins, a goal that could facilitate attempts to overexpress and/or refold them.
为了描述对整合膜蛋白折叠和组装过程的热力学限制，我们对细菌视紫红质（BR）的结构进行了生物物理剖析，BR是一种典型的α螺旋整合膜蛋白。合成了七条多肽，分别对应于BR中的七个跨膜α螺旋，并且在重构的磷脂囊泡中对每个单独多肽的结构进行了表征。七条多肽中的五条在与BR三级结构的其余部分分离时形成稳定的跨膜α螺旋。然而，使用我们的重构方案，BR中对应于F螺旋的多肽在重构囊泡中不形成任何稳定的二级结构，而对应于G螺旋的多肽形成超稳定的β折叠结构，其链垂直于膜平面定向。[对应于C螺旋的多肽以pH依赖的方式在跨膜α螺旋构象、外周结合的非螺旋构象和完全水溶性构象之间自发平衡；该多肽的构象特性是随附论文的主题：亨特等人（1997年）《生物化学》36，15177 - 15192。]我们的观察结果表明，α螺旋整合膜蛋白的折叠可能自发进行。然而，两条多肽表现出对非天然构象的偏好，这表明一些跨膜亚结构的形成可能需要外部限制，如螺旋之间的连接、与蛋白质其余部分的相互作用，或细胞伴侣或转位酶的参与。我们的结果还提出了一种提高α螺旋整合膜蛋白热力学稳定性的策略，这一目标可能有助于尝试对它们进行过量表达和/或重折叠。
