Detecting protein-protein interactions with a green fluorescent protein fragment reassembly trap: Scope and mechanism
Detecting protein-protein interactions with a green fluorescent protein fragment reassembly trap: Scope and mechanism
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DOI：
10.1021/ja046699g
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发表时间：
2005-01-12
影响因子：
15
通讯作者：
Regan, L
中科院分区：
文献类型：
--
作者：
Magliery, TJ;Wilson, CGM;Regan, L
蛋白质结合伙伴的确定是蛋白质组学的关键挑战之一。我们最近介绍了一种基于将绿色荧光蛋白的切割片段重组为相互作用的多肽来检测蛋白质-蛋白质相互作用的屏幕。在这里，我们提出了一组已获得的大肠杆菌质粒，用于方便地亚克隆与绿色荧光蛋白片段的融合。使用反平行亮氨酸拉链的库，我们已经表明，屏幕可以检测到非常弱的相互作用(K-D约为1毫米)。体外动力学表明，重组反应基本上是不可逆的，这表明该屏幕可能有助于检测瞬时相互作用。最后，我们使用该筛选来区分四肽重复结构域的同源和非同源的蛋白质-配体相互作用。这些实验证明了筛选较大蛋白质的一般效用，并阐明了机制细节，以指导在蛋白质组分析中进一步使用该筛选。此外，这项工作还揭示了反平行螺旋线圈中稳定相互作用的位置不等价性。
Identification of protein binding partners is one of the key challenges of proteomics. We recently introduced a screen for detecting protein-protein interactions based on reassembly of dissected fragments of green fluorescent protein fused to interacting peptides. Here, we present a set of comaintained Escherichia coli plasmids for the facile subcloning of fusions to the green fluorescent protein fragments. Using a library of antiparallel leucine zippers, we have shown that the screen can detect very weak interactions (K-D approximate to 1 mM). In vitro kinetics show that the reassembly reaction is essentially irreversible, suggesting that the screen may be useful for detecting transient interactions. Finally, we used the screen to discriminate cognate from noncognate protein-ligand interactions for tetratricopeptide repeat domains. These experiments demonstrate the general utility of the screen for larger proteins and elucidate mechanistic details to guide the further use of this screen in proteomic analysis. Additionally, this work gives insight into the positional inequivalence of stabilizing interactions in antiparallel coiled coils.