Photoaffinity labeling of heme proteins with chemically modified prosthetic groups

对于血红素和钼辅因子插入脱辅基蛋白的分析，基于化学的新工具是必不可少的。特别感兴趣的将是二氮丙啶功能化血红素类似物，将在与脱辅基蛋白的重建实验中进行分析，与我们在PROTRAIN研究组内的合作伙伴密切合作。关键的挑战是将二氮丙啶部分掺入辅因子或蛋白质氨基酸中，并具有最小的结构改变。最终，旨在通过释放反应性卡宾的二氮杂环丙烯部分的光解来中断重构。脱辅基蛋白将沿插入通道沿着官能化，并通过LTQ-轨道阱质谱法进行分析。血红素衍生物也将应用于寻找血红素结合蛋白，采用新的荧光和光活化连接。荧光三功能接头将被调查的工具，通过标签转移的蛋白质-蛋白质相互作用的鉴定。为了能够评估含有钼辅因子（Moco）的氧化还原酶的辅因子负荷，解决了Moco降解产物形式A和B的全合成，其特征在于具有挑战性的蝶啶衍生结构。

For the analysis of heme and molybdenum cofactor insertion into apoproteins novel chemistry-based tools are essential. Of particular interest will be diazirine-functionalized heme analogs that are to be analyzed in reconstitution experiments with apoproteins, in close collaboration with our partners within the PROTRAIN research group. Key challenge is the incorporation of diazirine moieties into cofactors or proteinogenic amino acids with minimal structural alteration. Ultimately, it is intended to interrupt the reconstitution by photolysis of the diazirine moieties liberating reactive carbenes. The apoproteins will be functionalized along the insertion channel and analyzed by LTQ-Orbitrap mass spectrometry. Heme derivatives will also be applied in the search for heme binding proteins employing novel fluorescent and photoactivatable linkers. Fluorescent trifunctional linkers will be investigated as tools for the identification of protein-protein interactions via label transfer. To be able to assess the cofactor load of redox enzymes containing the molybdenum cofactor (Moco) the total synthesis of the Moco degradation products Form A and B is addressed, which feature challenging pteridin-derived architectures.