Design of Cyborg Proteins by Loop Region Replacement with Oligo(ethylene glycol): Exploring Suitable Mutations for Cyborg Protein Construction Using Machine Learning

IF 3.3 3区化学 Q2 CHEMISTRY, MULTIDISCIPLINARY

Bulletin of the Chemical Society of Japan Pub Date : 2024-09-02 DOI:10.1093/bulcsj/uoae090

Wijak Yospanya, Akari Matsumura, Yukihiro Imasato, Tomoyuki Itou, Yusuke Aoki, Hikaru Nakazawa, Takashi Matsui, Takeshi Yokoyama, Mihoko Ui, Mitsuo Umetsu, Satoru Nagatoishi, Kouhei Tsumoto, Yoshikazu Tanaka, Kazushi Kinbara

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Abstract

We synthesized a “cyborg protein,” wherein a synthetic molecule partially substitutes the main peptide chain by linking two protein domains with a synthetic oligomer. Green fluorescent protein (GFP) served as the model for constructing the cyborg proteins. We prepared circularly permuted GFP (cpGFP) with new termini between β10 and β11, where the original N- and C-termini were linked by a cleavable peptide loop. The cyborg GFP was constructed from cpGFP by linking the β10 and β11 with oligo(ethylene glycol) using maleimide-cysteine couplings, followed by the enzymatic cleavage of the N- and C-termini linking loop by thrombin. With the help of machine learning, we were able to obtain the cpGFP mutants that significantly alter the fluorescence activity (53% increase) by thrombin treatment, which splits cpGFP into two fragments (fragmented-GFP), and by heat shock. When the cyborg GFP was constructed using this mutant, the fluorescence intensity increased by 13% after heat treatment, similar to cpGFP (33% increase), and the behavior was significantly different from that of the fragmented-GFP. This result suggests the possibility that the oligo(ethylene glycol) chain in the cyborg protein plays a similar role to the peptide in the main chain of the protein.

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用寡聚（乙二醇）取代环区设计半机械蛋白质：利用机器学习探索构建半机械蛋白质的合适突变

我们合成了一种 "半机械蛋白"，即用合成寡聚体连接两个蛋白质结构域，从而用合成分子部分取代主肽链。绿色荧光蛋白（GFP）是构建半机械蛋白的模型。我们制备了在 β10 和 β11 之间具有新末端的环状包覆 GFP（cpGFP），其中原来的 N 端和 C 端通过一个可裂解的肽环连接起来。我们利用马来酰亚胺-半胱氨酸偶联剂将β10和β11与寡聚（乙二醇）连接起来，然后用凝血酶酶解N端和C端的连接环，从而从cpGFP中构建出了半机械GFP。在机器学习的帮助下，我们通过凝血酶处理（凝血酶将 cpGFP 分裂成两个片段（fragmented-GFP））和热休克，获得了显著改变荧光活性（增加 53%）的 cpGFP 突变体。当使用这种突变体构建 cyborg GFP 时，热处理后荧光强度增加了 13%，与 cpGFP 相似（增加 33%），其行为与片段化 GFP 显著不同。这一结果表明，cyborg 蛋白中的寡聚（乙二醇）链可能起着类似于蛋白质主链中多肽的作用。

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来源期刊

Bulletin of the Chemical Society of Japan 化学-化学综合

CiteScore

6.40

自引率

5.00%

发文量

194

审稿时长

3-8 weeks

期刊介绍： The Bulletin of the Chemical Society of Japan (BCSJ) is devoted to the publication of scientific research papers in the fields of Theoretical and Physical Chemistry, Analytical and Inorganic Chemistry, Organic and Biological Chemistry, and Applied and Materials Chemistry. BCSJ appears as a monthly journal online and in advance with three kinds of papers (Accounts, Articles, and Short Articles) describing original research. The purpose of BCSJ is to select and publish the most important papers with the broadest significance to the chemistry community in general. The Chemical Society of Japan hopes all visitors will notice the usefulness of our journal and the abundance of topics, and welcomes more submissions from scientists all over the world.