Discovery of Light-Responsive Ligands through Screening of a Light-Responsive Genetically Encoded Library
Discovery of Light-Responsive Ligands through Screening of a Light-Responsive Genetically Encoded Library
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DOI：
10.1021/cb4006722
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发表时间：
2014-02-01
影响因子：
4
通讯作者：
Derda, Ratmir
中科院分区：
文献类型：
--
作者：
Jafari, Mohammad R.;Deng, Lu;Derda, Ratmir
光响应配体是生物化学和细胞生物学研究的重要工具，其功能具有空间和时间上的可调控性。这类配体的传统设计依赖于先前有关配体和受体结构的可用数据。在本文中，我们描述了通过筛选基因编码光响应文库的光响应配体的从头发现。我们将一个光响应偶氮苯核心连接到M13噬菌体外壳蛋白上随机显示的CX7C肽库上。半胱氨酸的一锅烷基化/还原产生了具有超过2 × 10(8)个成员的随机七肽大环的光响应文库。我们对噬菌体上的反应进行了表征，并优化了噬菌体文库中修饰物的产量。文库对链霉亲和素的筛选得到三个大环，它们在黑暗中与链霉亲和素结合，在370 nm光照射下停止结合。所有的配体在热松弛后都恢复了它们的结合特性，并且可以在几个周期内打开和关闭。我们用电喷雾电离质谱（ESI-MS）结合法测量解离常数K-d。对于配体ACGFERERTCG，顺式和反式异构体的K-d相差22倍；然而，不完全异构化（85%）导致暗态和辐照态之间的明显差异为4.5倍。我们期望本报告中描述的选择策略可以用于寻找许多没有已知天然配体的靶标的光响应配体。
Light-responsive ligands are useful tools in biochemistry and cell biology because the function of these ligands can be spatially and temporally controlled. Conventional design of such ligands relies on previously available data about the structure of both the ligand and the receptor. In this paper, we describe de novo discovery of light-responsive ligands through screening of a genetically encoded light-responsive library. We ligated a photoresponsive azobenzene core to a random CX7C peptide library displayed on the coat protein of M13 phage. A one-pot alkylation/reduction of the cysteines yielded a photoresponsive library of random heptapeptide macrocycles with over 2 X 10(8) members. We characterized the reaction on-phage and optimized the yield of the modifications in phage libraries. Screening of the library against streptavidin yielded three macrocycles that bind to streptavidin in the dark and cease binding upon irradiation with 370 nm light. All ligands restored their binding properties upon thermal relaxation and could be turned ON and OFF for several cycles. We measured dissociation constants, K-d, by electrospray ionization mass spectrometry (ESI-MS) binding assay. For ligand ACGFERERTCG, the K-d of cis and trans isomers differed by 22-fold; an incomplete isomerization (85%), however, resulted in the apparent difference of 4.5-fold between the dark and the irradiated state. We anticipate that the selection strategy described in this report can be used to find light-responsive ligands for many targets that do not have known natural ligands.