DiZPK Hydrochloride|安捷凯

Background

DiZPK Hydrochloride as a probe has two characteristics, the first DiZPK Hydrochloride as a derivative of pyrrolysine (the 22nd natural amino acid) can be used for genetic codon expansion technology; The non-natural amino acid can be introduced into the stop codon TAG position through the modification of pyrrole lyricyl-TrNA synthase (PylRS) and its corresponding tRNA(tRNAPyl) system. In addition, DiZPK Hydrochloride contains diazirine photocrosslinking group at specific 365 nm. Under wavelength irradiation, bisacproridine can be converted to free radical group, thus realizing the transient protein-protein non-covalent conversion to covalent action. Compared with other photocrosslinked groups, the "short" structure of the Linker with bisacridine can minimize the impact on the function of the target protein^[1].

DiZPK Hydrochloride revealed a crosslinking efficiency of 60% within 1 min of UV exposure and achieved a maximum efficiency higher than 80% within 3 min demonstrating the high flexibility of DiZPK^[2].

By site-specific introduction of DiZPK photoaffinity probes onto HdeA, it combines with gel-based proteomics to allow substrate analysis of HdeA in living E.coli cells at very low pH. Two important periplasmic partners, DegP and SurA, were found in the natural substrates identified by mass spectrometry, initially protected by HdeA at low pH, but subsequently assisting in HDEa-mediated refolding of other proteins after neutralization^[2].

参考文献:
[1]. 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.
[2]. 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作为探针有两个特点，第一DiZPK Hydrochloride作为吡咯赖氨酸（第22位天然氨基酸）的衍生物，可用于遗传密码子扩展技术；通过修饰吡咯赖氨酰-TrNA合酶(PylRS)及其相应的tRNA(tRNAPyl)系统，可以将非天然氨基酸引入终止密码子TAG位置。此外，DiZPK Hydrochloride 在特定的 365 nm 处含有二氮丙啶光交联基团。在波长照射下，双吖丙啶可转化为自由基团，从而实现瞬时蛋白质-蛋白质非共价转化为共价作用。与其他光交联基团相比，具有比吖啶的Linker的"短"结构可以最大限度地减少对目标蛋白功能的影响^[1]。

DiZPK Hydrochloride揭示了一种交联 structure of the Linker with bisacridine can minimize the impact on the function of the target protein^[1].

DiZPK Hydrochloride revealed a crosslinkingen_zh_2022q2.mdefficiency of 60% within 1 min of UV exposure and achieved a maximum efficiency higher than 80% within 3 min demonstrating the high flexibility of DiZPK^[2].

By site-specificen_zh_2022q2.mdintroduction of DiZPK photoaffinity probes onto HdeA, it combines with gel-based proteomics to allow substrate analysis of HdeA in living E.coli cells at very low pH.en_zh_2022q2.mdTwo important periplasmic partners, DegP and SurA, were found in the natural substrates identified by mass spectrometry, initially protected by HdeA at low pH, but subsequently assisting in HDEa-mediated refolding of other proteins after neutralization^[2].

Protocol

Assessing the crosslinking efficiency of DiZPK Hydrochloride [1]:

Preparation Method

Assessing the crosslinking efficiency of DiZPK Hydrochloride by analyzing the amounts of crosslinked products produced after they were introduced at a common position, Phe28, which is located at the dimer interface of the HdeA structure. The crosslinking was performed at neutral pH by exposing the E. coli cells, which heterologously expressed HdeA-His, and carried either of the two probes, to UV light (365 nm) for different time periods.The crosslinked dimer products of HdeA were then analyzed by SDS-PAGE and immunoblotting with a His-tag specific antibody.

Applications

For DiZPK Hydrochloride revealed a crosslinking efficiency of 60% within 1 min of UV exposure and achieved a maximum efficiency higher than 80% within 3 min demonstrating the higher flexibility of DiZPK.

DiZPK probe to capture periplasmic proteins interacting with HdeA in E. coli cells [2]:

Cell lines

E. coli cells

Preparation Method

Cells expressing HdeA proteins and carrying a singlesite incorporated DiZPK Hydrochloride at a variety of specific positions were grown at 37 ?校nd treated at pH 2.3 for 30 min before being subjected to 365nm light irradiation for 3 min. Immunoblotting analysis of lysates about the acid-treated cells.

Applications

DiZPK Hydrochloride was introduced into the two hydrophobic regions of HdeA, there were multiple protein bands containing HdeA. These bands showed higher molecular weight than the HdeA monomer and thus represented the cross-linked form of HdeA.

参考文献:

[1]. 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.

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DiZPK Hydrochloride

DiZPK Hydrochloride详情

Background

Protocol

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