Masuda Souta, Tomohiro Takenori, Yamaguchi Shouta, Morimoto Shota, Hatanaka Yasumaru
Laboratory of Biorecognition Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2603 Sugitani, Toyama 930-0194, Japan.
Laboratory of Biorecognition Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2603 Sugitani, Toyama 930-0194, Japan.
Bioorg Med Chem Lett. 2015 Apr 15;25(8):1675-1678. doi: 10.1016/j.bmcl.2015.03.008. Epub 2015 Mar 10.
Photoaffinity labeling (PAL) technique using a fluorogenic cross-linker is used to monitor the nucleotide-binding pocket within a protein. A coumarin fluorophore formed in the binding domain due to ultraviolet (UV) irradiation has been shown to accelerate the sequencing of the labeled peptide as well as identification of the labeled site by liquid chromatography (LC)-tandem mass spectrometry (MS), in addition to providing information on the ligand binding state. Selective monitoring of the predefined fluorescence peaks among the numerous digests obtained from high performance liquid chromatography (HPLC) clearly indicates the binding capability of the ligand to the entire protein as well as to the corresponding binding domain under various conditions. In the current study, ligand-binding analysis confirmed by the structural information of the binding state has been demonstrated using fluorogenic ATP/ADP photoactivatable probes under allosteric regulation of multiple substrates in the enzyme glutamate dehydrogenase (GDH).
使用荧光交联剂的光亲和标记（PAL）技术用于监测蛋白质内的核苷酸结合口袋。已证明，由于紫外线（UV）照射在结合域中形成的香豆素荧光团，除了提供有关配体结合状态的信息外，还能加速标记肽的测序以及通过液相色谱（LC）-串联质谱（MS）鉴定标记位点。在从高效液相色谱（HPLC）获得的众多消化产物中对预定义荧光峰进行选择性监测，清楚地表明了配体在各种条件下与整个蛋白质以及相应结合域的结合能力。在当前研究中，已使用荧光ATP/ADP光活化探针在谷氨酸脱氢酶（GDH）中多种底物的变构调节下，通过结合状态的结构信息证实了配体结合分析。
