Pfaffinger P J, DeRubeis D
Division of Neuroscience, Baylor College of Medicine, Houston, Texas 77030, USA.
J Biol Chem. 1995 Dec 1;270(48):28595-600. doi: 10.1074/jbc.270.48.28595.
The potassium channel T1 domain plays an important role in the regulated assembly of subunit proteins. We have examined the assembly properties of the Shaker channel T1 domain to determine if the domain can self-assemble, the number of subunits in a multimer, Ns and the mechanism of assembly. High pressure liquid chromatography (HPLC) size exclusion chromotography (SEC) separates T1 domain proteins into two peaks. By co-assembly assays, these peaks are identified to be a high molecular weight assembled form and a low molecular weight monomeric form. To determine the Ns of the assembled protein peak on HPLC SEC, we first cross-linked the T1 domain proteins and then separated them on HPLC. Four evenly spaced bands co-migrate with the assembled protein peak; thus, the T1 domain assembles to form a tetramer. The absence of separate dimeric and trimeric peaks of assembled T1 domain protein suggests that the tetramer is the stable assembled state, most probably a closed ring structure.
钾通道T1结构域在亚基蛋白的调控组装中起重要作用。我们研究了Shaker通道T1结构域的组装特性，以确定该结构域是否能自我组装、多聚体中亚基的数量Ns以及组装机制。高压液相色谱（HPLC）尺寸排阻色谱（SEC）将T1结构域蛋白分离为两个峰。通过共组装分析，这些峰被鉴定为高分子量组装形式和低分子量单体形式。为了确定HPLC SEC上组装蛋白峰的Ns，我们首先交联T1结构域蛋白，然后在HPLC上分离它们。四条等间距的条带与组装蛋白峰共同迁移；因此，T1结构域组装形成四聚体。组装的T1结构域蛋白不存在单独的二聚体和三聚体峰，这表明四聚体是稳定的组装状态，很可能是闭环结构。
