| 研究課題/領域番号 |
24K21927
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| 研究種目 |
挑戦的研究(萌芽)
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| 配分区分 | 基金 |
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| 審査区分 |
中区分43:分子レベルから細胞レベルの生物学およびその関連分野
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| 研究機関 | 東京科学大学 |
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研究代表者 |
ロンゴ リアム 東京科学大学, 未来社会創成研究院, 特任准教授 (40955027)
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| 研究分担者 |
松浦 友亮 東京科学大学, 地球生命研究所, 教授 (50362653)
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| 研究期間 (年度) |
2024-06-28 – 2026-03-31
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| 研究課題ステータス |
交付 (2024年度)
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| 配分額 *注記 |
6,500千円 (直接経費: 5,000千円、間接経費: 1,500千円)
2025年度: 3,250千円 (直接経費: 2,500千円、間接経費: 750千円)
2024年度: 3,250千円 (直接経費: 2,500千円、間接経費: 750千円)
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| キーワード | transporter / EmrE / protein evolution / evolvability / sequence reconstruction / functional promiscuity |
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| 研究開始時の研究の概要 |
Ancestral sequences are thought to be multifunctional, allowing them to operate on diverse substrates. In other words, ancestral enzymes with multifunctionality are thought to have evolved into extant enzymes with high specificity. Multidrug efflux transporters, on the other hand, exist in all three domains of life and can transport drugs with diverse chemical structures. Existing multidrug efflux transporters retain the multifunctionality that enzymes often lose during evolution. What kind of evolutionary process did multifunctional proteins go through to arrive at their modern properties?
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| 研究実績の概要 |
Phylogenetic analysis and ancestral sequence reconstruction (ASR) were performed using standard approaches on the EmrE transporter family. HMM profiles for the EmrE transporter family were used to collect homologs and sequences were clustered (CD-HIT), aligned (MAFFT), trimmed (trimAl), phylogenetic tree calculated (FastTree, IQ-TREE), and ancestor sequences calculated (codeML). Genes for 5 of the ancestors from the EmrE tree were selected synthesized. E. coli cell lines harboring these genes were subject to cell growth assays in the presence of antibiotics to confirm the in vivo activity of the transporters. Ancestral sequences were found to be active. A cell-free assay for EmrE active production was developed using the PURE system and the chaperone MPIase. The activity of the in vitro-produced proteins was analyzed by a radioactive transport assay that was developed as part of the project. Taken together, these data demonstrate the feasibility of analyzing the functional trajectory of EmrE proteins over evolutionary time.
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| 現在までの達成度 |
現在までの達成度
2: おおむね順調に進展している
理由
The current progress on the project is satisfactory. The bioinformatic aspect of the project has been complete on time. Currently, both in vivo and in vitro assays for testing the activity of EmrE variants, including the ancestors, have been developed. Currently, purification protocols and the development of SSME protocols are underway. Already, the activity of the ancestors has been shown. This progress is in line with the predicted project timeline.
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| 今後の研究の推進方策 |
In addition to the proposed research outlines, this project has uncovered the role of membrane chaperones (MPIase) in meditating the insertion of EmrE into the membrane and adopting a functional conformation. Thus, the importance of this membrane chaperone will be analyzed in further detail for both its biological role and its utility for biotechnological applications. However, this does not require a significant departure from the proposed research plan.
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