Transmembrane Domain Oligomerization Propensity determined by ToxR Assay
Transmembrane Domain Oligomerization Propensity determined by ToxR Assay
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DOI：
10.3791/2721
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发表时间：
2011-05-01
影响因子：
1.2
通讯作者：
Yin, Hang
中科院分区：
文献类型：
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作者：
Joce, Catherine;Wiener, Alyssa;Yin, Hang
蛋白质跨膜结构域仅仅是磷脂双分子层中的锚的过于简单的观点早已被证明是错误的。在许多情况下，跨膜蛋白已经进化出高度复杂的作用机制。(1-3)膜蛋白调节其结构和功能的一种方式是通过疏水性螺旋的直接和特异性接触，形成结构化的跨膜寡聚体。（4，5）最近的许多工作集中在与水溶液相比优先在膜环境中发现的氨基酸的分布以及驱动蛋白质缔合的不同分子间力。（6，7）然而，蛋白质跨膜结构域的分子识别研究仍然落后于水溶性区域的研究。一个主要障碍仍然存在：尽管跨膜寡聚化可以实现显著的特异性和亲和力，（8）直接测量它们的结合是具有挑战性的。传统的方法应用于研究膜蛋白的功能，可以阻碍固有的不溶性的序列检查。从研究代表跨膜结构域的合成肽中获得的生物物理学见解可以提供有用的结构见解。然而，在这些研究中用于模拟细胞膜的洗涤剂胶束或脂质体系统的生物相关性经常受到质疑;肽在这些条件下是否采用天然样结构，其功能行为是否真实地反映了天然膜内的作用模式？为了研究天然磷脂双层中跨膜序列的相互作用，Langosch实验室开发了ToxR转录报告基因测定。(9)目的跨膜结构域表达为具有麦芽糖结合蛋白的嵌合蛋白，用于定位于周质和ToxR，以提供寡聚化水平的报告（图1）。（10-13）各种ToxR测定已成为测试细胞膜中蛋白质-蛋白质相互作用的金标准。我们在这里展示了一个典型的实验操作，在我们的实验室进行，主要遵循由Langosch开发的协议。该方法可用于分析大肠杆菌跨膜结构域的自结合。大肠杆菌，其中β-半乳糖苷酶的产生用于评估TMD寡聚化倾向。在TMD诱导的二聚化后，ToxR与ctx启动子结合，导致β-半乳糖苷酶的LacZ基因上调。通过向裂解细胞中加入ONPG获得比色读数。通过β-半乳糖苷酶水解ONPG导致产生光吸收物质邻硝基苯酚盐（ONP）（图2）。
The oversimplified view of protein transmembrane domains as merely anchors in phospholipid bilayers has long since been disproven. In many cases membrane-spanning proteins have evolved highly sophisticated mechanisms of action. (1-3) One way in which membrane proteins can modulate their structures and functions is by direct and specific contact of hydrophobic helices, forming structured transmembrane oligomers. (4,5) Much recent work has focused on the distribution of amino acids preferentially found in the membrane environment in comparison to aqueous solution and the different intermolecular forces that drive protein association. (6,7) Nevertheless, studies of molecular recognition at the transmembrane domain of proteins still lags behind those of water-soluble regions. A major hurdle remains: despite the remarkable specificity and affinity that transmembrane oligomerization can achieve, (8) direct measurement of their association is challenging. Traditional methodologies applied to the study of integral membrane protein function can be hampered by the inherent insolubility of the sequences under examination. Biophysical insights gained from studying synthetic peptides representing transmembrane domains can provide useful structural insight. However, the biological relevance of the detergent micellar or liposome systems used in these studies to mimic cellular membranes is often questioned; do peptides adopt a native-like structure under these conditions and does their functional behaviour truly reflect the mode of action within a native membrane? In order to study the interactions of transmembrane sequences in natural phospholipid bilayers, the Langosch lab developed ToxR transcriptional reporter assays. (9) The transmembrane domain of interest is expressed as a chimeric protein with maltose binding protein for location to the periplasm and ToxR to provide a report of the level of oligomerization (Figure 1).In the last decade, several other groups (e.g. Engelman, DeGrado, Shai) further optimized and applied this ToxR reporter assay. (10-13) The various ToxR assays have become a gold standard to test protein-protein interactions in cell membranes. We herein demonstrate a typical experimental operation conducted in our laboratory that primarily follows protocols developed by Langosch. This generally applicable method is useful for the analysis of transmembrane domain self-association in E. coli, where beta-galactosidase production is used to assess the TMD oligomerization propensity. Upon TMD-induced dimerization, ToxR binds to the ctx promoter causing up-regulation of the LacZ gene for beta-galactosidase. A colorimetric readout is obtained by addition of ONPG to lyzed cells. Hydrolytic cleavage of ONPG by beta-galactosidase results in the production of the light absorbing species o-nitrophenolate (ONP) (Figure 2).