Strategic Amino Acid Mutations in CPD Cleavage Motif: Impacts on Hydrolysis and C-Terminal Modification Efficiency
文献类型：期刊论文
| 作者 | Yashinov, Ansor2,3; Zou, Xiangman2,6; Hang, Jiayin2,3; Liu, Zhi1,5; Song, Fengnan4; Zeng, Yue2; Yang, Yang4; Xia, Fei2; Tang, Feng2; Shi, Wei2 |
| 刊名 | CHEMICAL RESEARCH IN CHINESE UNIVERSITIES
 |
| 出版日期 | 2025-06-01 |
| 卷号 | 41期号:3页码:592-600 |
| 关键词 | Protein engineering C-Terminal modification Cysteine protease domain Self-cleavage tag AlphaFold2 |
| ISSN号 | 1005-9040 |
| DOI | 10.1007/s40242-025-5013-0 |
| 英文摘要 | Precise modification of the C-terminus of proteins is crucial for investigating protein-protein interaction and enhancing protein functionalities. While traditional methods face challenges due to multiple reactive sites, recent advancements have introduced cysteine protease domain (CPD) tag for efficient C-terminal modifications. CPD, when fused with proteins of interest (POI), can facilitate concurrent hydrolysis and amidation under Inositol hexakisphosphate (InsP6) activation. Herein, we explored the influence of substituting the Ala residue following Leu in the CPD cleavage motif (VDALADGK) with each of the 19 other amino acids. By creating a series of green fluorescent protein (GFP)-CPD fusion constructs, we evaluated their hydrolysis and amidation efficiencies. Our results revealed that mutations to Ser and Asn significantly enhanced C-terminal modification, while Pro substitution completely hindered hydrolysis activity. Additionally, we demonstrated the successful labeling of a Ser mutant with a fluorescent probe, establishing its potential for F & ouml;rster resonance energy transfer (FRET) applications. Structural analyses using AlphaFold2 indicated that the observed variations in activity could be attributed to the differences in molecular interactions and the flexibility of the substituted amino acids. Overall, this research highlights the utility of strategically designed mutations in enhancing C-terminal modifications, offering valuable insights for future protein engineering endeavors. |
| WOS关键词 | ALPHAFOLD2 ; PROTEINS ; FUNCTIONALIZATION ; ACTIVATION |
| 资助项目 | National Science Fund for Distinguished Young Scholars, China[82325045] ; National Natural Science Foundation of China[82204183] ; National Natural Science Foundation of China[22277126] ; Shanghai Sail Program, China[22YF1457400] ; Hangzhou Innovation and Entrepreneurship Leading Team Project, China[TD2020005] |
| WOS研究方向 | Chemistry |
| 语种 | 英语 |
| WOS记录号 | WOS:001483833600001 |
| 出版者 | HIGHER EDUCATION PRESS |
| 源URL | [http://119.78.100.183/handle/2S10ELR8/317590]  |
| 专题 | 新药研究国家重点实验室 |
| 通讯作者 | Shi, Wei; Huang, Wei |
| 作者单位 | 1.Shanghai Tech Univ, Sch Phys Sci & Technol, Shanghai 201210, Peoples R China 2.Chinese Acad Sci, Shanghai Inst Mat Med, State Key Lab Drug Res, Shanghai 201203, Peoples R China 3.Univ Chinese Acad Sci, Beijing 100049, Peoples R China 4.Hangzhou Inst Adv Study, Sch Pharmaceut Sci & Technol, Hangzhou 310024, Peoples R China 5.Lingang Lab, Shanghai 200031, Peoples R China 6.Shenyang Pharmaceut Univ, Key Lab Struct Based Drug Design & Discovery, Minist Educ, Shenyang 110016, Peoples R China 7.Shanghai GlycanLink Biotech Co Ltd, Shanghai 201210, Peoples R China |
| 推荐引用方式 GB/T 7714 | Yashinov, Ansor,Zou, Xiangman,Hang, Jiayin,et al. Strategic Amino Acid Mutations in CPD Cleavage Motif: Impacts on Hydrolysis and C-Terminal Modification Efficiency[J]. CHEMICAL RESEARCH IN CHINESE UNIVERSITIES,2025,41(3):592-600. |
| APA | Yashinov, Ansor.,Zou, Xiangman.,Hang, Jiayin.,Liu, Zhi.,Song, Fengnan.,...&Huang, Wei.(2025).Strategic Amino Acid Mutations in CPD Cleavage Motif: Impacts on Hydrolysis and C-Terminal Modification Efficiency.CHEMICAL RESEARCH IN CHINESE UNIVERSITIES,41(3),592-600. |
| MLA | Yashinov, Ansor,et al."Strategic Amino Acid Mutations in CPD Cleavage Motif: Impacts on Hydrolysis and C-Terminal Modification Efficiency".CHEMICAL RESEARCH IN CHINESE UNIVERSITIES 41.3(2025):592-600. |
入库方式： OAI收割
来源：上海药物研究所
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