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Mapping of protein-peptide and protein-protein interactions by means of genetically encoded photocrosslinkers

通过基因编码的光交联剂绘制蛋白质-肽和蛋白质-蛋白质相互作用的图谱

基本信息

批准号：
236346005
负责人：
Professorin Dr. Irene Coin
金额：
--
依托单位：
Arbeitsgruppe Synthetische Proteinbiochemie
依托单位国家：
德国
项目类别：
Independent Junior Research Groups
财政年份：
2013
资助国家：
德国
起止时间：
2012-12-31 至 2019-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/236346005?language=en
关键词：
Mapping protein peptide interactions means

项目摘要

The project aims at establishing a general methodology to investigate peptide-protein and protein- protein interactions in intact mammalian cells based on the use of unnatural amino acid mutagenesis and photoaffinity crosslinking. This will provide a tool for mapping the topology of interaction sites as well as for searching for unknown interactions between proteins. Our primary molecular target is a membrane protein, the class-B GPCR c orticotropin releasing factor receptor type 1 (CRFR1), which represents the key element of the organism response to stress stimuli. We will use libraries of CRFR1 mutants bearing a single photoactivatable amino acid, p-azido-Phe (Azi), at each position of selected domains to test the ability of each site to capture peptide ligands. In this way we have very recently located the J-domain binding pocket of CRFR1 for the native polypeptide agonist Ucn-I. In project A we will build crosslinking maps of peptide ligands showing different patterns of Gs/Gi activation. In parallel, we will study how interaction sites within the CRFR1-Ucn-I complex change, when the receptor is pre-coupled to specific G-proteins. This will give first information about molecular determinants that trigger different signaling pathways. Project B aims at characterizing the site of crosslinking in the pray molecule at the single amino acids level. First, we will use the azide-alkyne click reaction to achieve site specific chemical crosslinking between Azi residues in the mutant receptors and propargylglycine residues incorporated at different positions of the ligand. In parallel, we will develop a method to purify crosslinked receptors based on the use of a Flag tag paired to a Biotin tag. The latter will be introduced posttranslationally through a transpeptidation reaction catalyzed by Sortase A. Optimized steps of enzymatic digestion will provide crosslinked fragments of low molecular weight to be analyzed with MS/MS. This will allow determining the ligand orientation in the binding pocket and build the first detailed binding model. In project C we will incorporate amino acids bearing benzophenone and diazirine photocrosslinkers into lipid-exposed positions of the transmembrane domains of CRFR1, to investigate the surfaces involved in the formation of homo-dimers. The same receptor mutants will serve as baits to capture other proteins interacting within the transmembrane region, a task that cannot be accomplished with any other currently available method. Crosslinked products will be enriched with the strategy developed in project B and associated proteins will be identified with mass spectrometry. The same approach will be used to map the interaction of type 2 CRFR with the oncoprotein ErbB2, and in the long term to investigate other interactions taking place at the intracellular domains. In perspective the methodology will allow investigating any protein-protein interaction involved in signaling cascades and other cellular processes in the native environment of the live cell.

该项目旨在建立一种基于非自然氨基酸诱变和光亲和交联的方法来研究完整哺乳动物细胞中肽-蛋白和蛋白-蛋白相互作用。这将为绘制相互作用位点的拓扑结构以及寻找蛋白质之间未知的相互作用提供工具。我们的主要分子靶点是一种膜蛋白，b类GPCR c促肾上腺皮质激素释放因子受体1型（CRFR1），它代表了生物体对应激刺激反应的关键因素。我们将使用CRFR1突变体文库，在选择的结构域的每个位置上携带单个光激活氨基酸，p-叠氮多-苯丙氨酸（Azi），以测试每个位点捕获肽配体的能力。通过这种方式，我们最近找到了天然多肽激动剂Ucn-I的CRFR1的j结构域结合袋。在项目A中，我们将构建显示不同Gs/Gi激活模式的肽配体的交联图。同时，我们将研究当受体预偶联到特定的g蛋白时，CRFR1-Ucn-I复合体内的相互作用位点是如何变化的。这将提供关于触发不同信号通路的分子决定因素的第一手信息。项目B旨在在单氨基酸水平上表征祈祷分子中交联的位点。首先，我们将使用叠氮化物-炔点击反应来实现突变受体中的叠氮化物残基与配体不同位置上的丙基甘氨酸残基之间的位点特异性化学交联。同时，我们将开发一种基于使用与生物素标签配对的Flag标签来纯化交联受体的方法。后者将通过Sortase a催化的转肽化反应在翻译后引入，优化的酶切步骤将提供低分子量的交联片段，用于MS/MS分析。这将允许确定结合口袋中的配体方向，并建立第一个详细的结合模型。在C项目中，我们将把含二苯甲酮和重氮嘧啶光交联剂的氨基酸纳入CRFR1跨膜结构域的脂质暴露位置，以研究参与形成同型二聚体的表面。相同的受体突变体将作为捕获跨膜区域内相互作用的其他蛋白质的诱饵，这是目前任何其他可用方法都无法完成的任务。交联产物将通过项目B中开发的策略进行富集，相关蛋白将通过质谱法进行鉴定。同样的方法将用于绘制2型CRFR与癌蛋白ErbB2的相互作用，并在长期内研究发生在细胞内结构域的其他相互作用。从长远来看，该方法将允许在活细胞的天然环境中研究任何涉及信号级联反应和其他细胞过程的蛋白质-蛋白质相互作用。