Endoproteolysis of Oligopeptide-Based Coacervates for Enzymatic Modeling,ACS Nano

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Endoproteolysis of Oligopeptide-Based Coacervates for Enzymatic Modeling
ACS Nano ( IF 15.8 ) Pub Date : 2023-08-14 , DOI: 10.1021/acsnano.3c04259
Zhicheng Jin ₁ , Chuxuan Ling ₁ , Wonjun Yim ₂ , Yu-Ci Chang ₂ , Tengyu He ₂ , Ke Li ₃ , Jiajing Zhou ₁ , Yong Cheng ₁ , Yi Li ₁ , Justin Yeung ₄ , Ruijia Wang ₅ , Pavla Fajtová ₆ , Lubna Amer ₂ , Hedi Mattoussi ₇ , Anthony J O'Donoghue ₆ , Jesse V Jokerst _{1,

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Affiliation

Better insights into the fate of membraneless organelles could strengthen the understanding of the transition from prebiotic components to multicellular organisms. Compartmentalized enzyme reactions in a synthetic coacervate have been investigated, yet there remains a gap in understanding the enzyme interactions with coacervate as a substrate hub. Here, we study how the molecularly crowded nature of the coacervate affects the interactions of the embedded substrate with a protease. We design oligopeptide-based coacervates that comprise an anionic Asp-peptide (D₁₀) and a cationic Arg-peptide (R₅R₅) with a proteolytic cleavage site. The coacervates dissolve in the presence of the main protease (M^pro) implicated in the coronavirus lifecycle. We capitalize on the condensed structure, introduce a self-quenching mechanism, and model the enzyme kinetics by using Cy5.5-labeled peptides. The determined specificity constant (k_cat/K_M) is 5817 M^–1 s^–1 and is similar to that of the free substrate. We further show that the enzyme kinetics depend on the type and quantity of dye incorporated into the coacervates. Our work presents a simple design for enzyme-responsive coacervates and provides insights into the interactions between the enzyme and coacervates as a whole.

中文翻译：

用于酶促建模的基于寡肽的凝聚层的内蛋白水解

更好地了解无膜细胞器的命运可以加强对从益生元成分到多细胞生物转变的理解。已经研究了合成凝聚层中的区室化酶反应，但在理解酶与凝聚层作为底物中心的相互作用方面仍然存在差距。在这里，我们研究了凝聚层的分子拥挤性质如何影响嵌入的底物与蛋白酶的相互作用。我们设计了基于寡肽的凝聚层，其包含阴离子天冬氨酸肽（D ₁₀ ）和带有蛋白水解切割位点的阳离子精氨酸肽（R ₅ R ₅ ）。凝聚层在冠状病毒生命周期中涉及的主要蛋白酶 (M ^pro ) 的存在下溶解。我们利用缩合结构，引入自猝灭机制，并使用 Cy5.5 标记的肽对酶动力学进行建模。确定的特异性常数 ( k _cat /K _M ) 为 5817 M ^–1 s ^–1 ，与游离底物的相似。我们进一步表明，酶动力学取决于掺入凝聚层中的染料的类型和数量。我们的工作提出了酶响应凝聚层的简单设计，并提供了对酶和凝聚层之间相互作用的见解。

更新日期：2023-08-14

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