Retention of native-like oligomerization states in transmembrane segment peptides:: Application to the Escherichia coli aspartate receptor
Retention of native-like oligomerization states in transmembrane segment peptides:: Application to the Escherichia coli aspartate receptor
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DOI：
10.1021/bi010642e
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发表时间：
2001-09-18
期刊：
影响因子：
2.9
通讯作者：
Deber, CM
中科院分区：
文献类型：
--
作者：
Melnyk, RA;Partridge, AW;Deber, CM
整体膜蛋白的跨膜（TM）结构域的生物物理研究一直受到其疏水性的阻碍。因此，对膜内蛋白质相互作用的细节的理解往往是缺乏的。我们之前已经证明，具有侧侧阳离子残基的模型TM片段在插入到膜模拟环境中时会自发折叠成α -螺旋。在这里，我们扩展了这些研究，以研究这些由生物系统中的TM螺旋组成的结构是否保留其天然的二级结构和低聚物状态。设计并合成了表皮生长因子受体（EGFR）、糖蛋白A （GPA）和甲型流感病毒M2离子通道（M2）的单跨TM结构域，并在N端和c端分别含有3 ~ 4个赖氨酸残基。每个结构体在插入十二烷基硫酸钠胶束后均显示为x-螺旋构象。此外，胶束插入的TM片段根据其各自的天然低聚物状态与SDS-PAGE凝胶相关联：EGFR为单体，GPA为二聚体，M2为四聚体。然后，该方法用于研究大肠杆菌天冬氨酸受体的TM片段（Tar-1和Tar-2）是否与其同型二聚体性质有关。结果表明，Tar-1形成抗sds的同二聚体，而Tar-2为单体。此外，在Tar-1和Tar-2之间没有检测到异齐聚，这意味着Tar-1螺旋是Tar蛋白的寡聚决定因素。总体结果表明，该方法可用于阐明单跨和多跨膜蛋白的TM结构域折叠细节。
Biophysical study of the transmembrane (TM) domains of integral membrane proteins has traditionally been impeded by their hydrophobic nature. As a result, an understanding of the details of protein-protein interactions within membranes is often lacking. We have demonstrated previously that model TM segments with flanking cationic residues spontaneously fold into alpha -helices upon insertion into membrane-mimetic environments. Here, we extend these studies to investigate whether such constructs consisting of TM helices from biological systems retain their native secondary structures and oligomeric states. Single-spanning TM domains from the epidermal growth factor receptor (EGFR), glycophorin A (GPA), and the influenza A virus M2 ion channel (M2) were designed and synthesized with three to four lysine residues at both N- and C-termini. Each construct was shown to adopt an (x-helical conformation upon insertion into sodium dodecyl sulfate micelles. Furthermore, micelle-inserted TM segments associated on SDS-PAGE gels according to their respective native-like oligomeric states: EGFR was monomeric, GPA was dimeric, and M2 was tetrameric. This approach was then used to investigate whether one or both of the TM segments (Tar-1 and Tar-2) from the Escherichia coli aspartate receptor were responsible for its homodimeric nature. Our results showed that Tar-1 formed SDS-resistant homodimers, while Tar-2 was monomeric. Furthermore, no heterooligomerization between Tar-1 and Tar-2 was detected, implicating the Tar-1 helix as the oligomeric determinant for the Tar protein. The overall results indicate that this approach can be used to elucidate the details of TM domain folding for both single-spanning and multispanning membrane proteins.