Organoruthenium-catalyzed chemical protein synthesis to elucidate the functions of epigenetic modifications on heterochromatin factors.
Organoruthenium-catalyzed chemical protein synthesis to elucidate the functions of epigenetic modifications on heterochromatin factors.
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DOI：
10.1039/d1sc00731a
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发表时间：
2021-04-28
期刊：
影响因子：
8.4
通讯作者：
Okamoto A
中科院分区：
文献类型：
--
作者：
Kamo N;Kujirai T;Kurumizaka H;Murakami H;Hayashi G;Okamoto A
金属有机化合物在蛋白质科学中的应用已受到关注。最近，已经开发了使用过渡金属络合物的全化学蛋白质合成以产生具有位点特异性翻译后修饰（PTM）的各种蛋白质。然而，一般来说，需要大量的金属络合物来实现具有大量亲核官能团的蛋白质的化学反应。此外，由于繁琐的纯化和分离步骤，中等大小蛋白质（>20 kDa）的合成受到耗时程序的困扰，这妨碍了获得各种修饰的蛋白质。在这里，我们报告了一锅多肽连接策略辅助的耐空气的有机钌催化剂，显示超过50倍的活性超过以前的钯络合物，导致快速和定量脱保护蛋白质与催化量（20摩尔%）的金属络合物，即使在存在过量的硫醇部分。利用有机钌催化剂，已经合成了20 kDa以上的异染色质因子，例如连接体组蛋白H1.2和异染色质蛋白1α（HP1α），其具有位点特异性的PTM，包括磷酸化、泛素化、瓜氨酸酶和乙酰化。使用合成蛋白质的生物化学测定显示，H1.2中R53处的瓜氨酸导致与DNA的静电相互作用降低，与核小体的结合亲和力降低。此外，我们确定了HP1α中控制其DNA结合能力的关键磷酸化区域。这里开发的钌化学将促进含有PTM和非天然氨基酸的各种生物学和医学上重要的蛋白质的制备。有机钌催化剂辅助的化学蛋白质合成简化了异染色质因子的产生，这些异染色质因子具有各种表观遗传修饰模式，并阐明了它们的生物学意义。
The application of organometallic compounds for protein science has received attention. Recently, total chemical protein synthesis using transition metal complexes has been developed to produce various proteins bearing site-specific posttranslational modifications (PTMs). However, in general, significant amounts of metal complexes were required to achieve chemical reactions of proteins bearing a large number of nucleophilic functional groups. Moreover, syntheses of medium-size proteins (>20 kDa) were plagued by time-consuming procedures due to cumbersome purification and isolation steps, which prevented access to variously decorated proteins. Here, we report a one-pot multiple peptide ligation strategy assisted by an air-tolerant organoruthenium catalyst that showed more than 50-fold activity over previous palladium complexes, leading to rapid and quantitative deprotection on a protein with a catalytic amount (20 mol%) of the metal complex even in the presence of excess thiol moieties. Utilizing the organoruthenium catalyst, heterochromatin factors above 20 kDa, such as linker histone H1.2 and heterochromatin protein 1α (HP1α), bearing site-specific PTMs including phosphorylation, ubiquitination, citrullination, and acetylation have been synthesized. The biochemical assays using synthetic proteins revealed that the citrullination at R53 in H1.2 resulted in the reduced electrostatic interaction with DNA and the reduced binding affinity to nucleosomes. Furthermore, we identified a key phosphorylation region in HP1α to control its DNA-binding ability. The ruthenium chemistry developed here will facilitate the preparation of a variety of biologically and medically significant proteins containing PTMs and non-natural amino acids. Chemical protein synthesis assisted by an organoruthenium catalyst streamlined the production of heterochromatin factors bearing various patterns of epigenetic modifications, and their biological significance was elucidated.
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影响因子：
14.9
作者：
Harshman SW;Young NL;Parthun MR;Freitas MA
通讯作者：
Freitas MA
H3K9ME3依赖性异染色质：细胞命运变化的屏障。
DOI：
10.1016/j.tig.2015.11.001
发表时间：
2016-01
期刊：
Trends in genetics : TIG
影响因子：
--
作者：
Becker JS;Nicetto D;Zaret KS
通讯作者：
Zaret KS
影响因子：
19
作者：
Canzio, Daniele;Larson, Adam;Narlikar, Geeta J.
通讯作者：
Narlikar, Geeta J.
影响因子：
56.9
作者：
DAWSON, PE;MUIR, TW;KENT, SBH
通讯作者：
KENT, SBH
影响因子：
3.2
作者：
Jbara, Muhammad;Eid, Emad;Brik, Ashraf
通讯作者：
Brik, Ashraf