DiZPK Hydrochloride is a photoreactive lysine analog containing a photoactivated diaziridine functional group that can promote covalent photocrosslinking of protein-protein interactions in prokaryotic and eukaryotic cells[1].
DiZPK Hydrochloride has two characteristics as a probe: first, as a derivative of pyrrolysine, DiZPK Hydrochloride can be used in genetic codon expansion technology; second, by modifying the pyrrolysine-TrNA synthase (PylRS) and its corresponding tRNA (tRNAPyl) system, non-natural amino acids can be introduced into the stop codon TAG position. In addition, DiZPK Hydrochloride contains a diacridine photocrosslinking group. Under specific 365nm wavelength irradiation, diacridine can be converted into free radical groups, thereby achieving transient protein-protein non-covalent conversion to covalent interaction. Compared with other photocrosslinking groups, the "short" structure of the diacridine linker can minimize the impact on the function of the target protein[2].
DiZPK Hydrochloride achieved a cross-linking efficiency of 60% within 1 minute of UV irradiation and a maximum efficiency of over 80% within 3 minutes, indicating that DiZPK has high flexibility[3].
By introducing the DiZPK photoaffinity probe site-specifically into HdeA, it was combined with gel-based proteomics to enable substrate analysis of HdeA in living Escherichia coli cells at very low pH. Two important periplasmic partners, DegP and SurA, were found among the natural substrates identified by mass spectrometry, which were initially protected by HdeA at low pH but then assisted the refolding of other proteins mediated by HDEa after neutralization[3].
References:
[1]. Chengjie Chen, Tao Peng.Protocol for Site-Specific Photo-Crosslinking Proteomics to Identify Protein-Protein Interactions in Mammalian Cells.STAR Protoc.2020 Sep15;1(3):100109.doi: 10.1016/j.xpro.2020.100109. eCollection 2020 Dec 18.
[2]. He D, Xie X, et,al. Quantitative and Comparative Profiling of Protease Substrates through a Genetically Encoded Multifunctional Photocrosslinker. Angew Chem Int Ed Engl. 2017 Nov 13;56(46):14521-14525. doi: 10.1002/anie.201708151. Epub 2017 Oct 11. PMID: 28940571.
[3]. Zhang M, Lin S, et,al. A genetically incorporated crosslinker reveals chaperone cooperation in acid resistance. Nat Chem Biol. 2011 Sep 4;7(10):671-7. doi: 10.1038/nchembio.644. PMID: 21892184.
DiZPK Hydrochloride是一种光反应性赖氨酸类似物，含有光激活的二吖丙啶官能团，能够促进原核和真核细胞中蛋白质-蛋白质相互作用的共价光交联[1]。
DiZPK Hydrochloride作为探针具有两个特点：其一，DiZPK Hydrochloride作为吡咯赖氨酸的衍生物，可用于遗传密码子扩展技术；其二，通过对吡咯赖氨酸-TrNA合酶（PylRS）及其对应的tRNA（tRNAPyl）系统的改造，可将非天然氨基酸引入终止密码子TAG位置。此外，DiZPK Hydrochloride含有双吖啶光交联基团，在特定的365nm波长照射下，双吖啶可转化为自由基基团，从而实现瞬时蛋白-蛋白非共价转化为共价作用。与其他光交联基团相比，双吖啶Linker的“短”结构可最大程度地减少对靶蛋白功能的影响[2]。
DiZPK Hydrochloride在紫外线照射1分钟内交联效率达到60%，3分钟内达到最高效率 80%以上，表明DiZPK具有很高的灵活性[3]。
通过将DiZPK光亲和探针位点特异性引入HdeA，它与基于凝胶的蛋白质组学相结合，可以在极低pH值下对活大肠杆菌细胞中的HdeA进行底物分析。在质谱鉴定的天然底物中发现了两个重要的周质伙伴DegP和SurA，它们最初在低pH值下受到HdeA的保护，但随后在中和后协助HDEa介导的其他蛋白质的重新折叠[3]。