Domain Shuffling and Site-Saturation Mutagenesis for the Enhanced Inhibitory Potential of Amaranthaceae α-Amylase Inhibitors

IF 1.9 4区 生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY The Protein Journal Pub Date : 2023-08-19 DOI:10.1007/s10930-023-10148-y
Ashwini S. Rane, Vineetkumar S. Nair, Rakesh S. Joshi, Ashok P. Giri
{"title":"Domain Shuffling and Site-Saturation Mutagenesis for the Enhanced Inhibitory Potential of Amaranthaceae α-Amylase Inhibitors","authors":"Ashwini S. Rane,&nbsp;Vineetkumar S. Nair,&nbsp;Rakesh S. Joshi,&nbsp;Ashok P. Giri","doi":"10.1007/s10930-023-10148-y","DOIUrl":null,"url":null,"abstract":"<div><p>Amaranthaceae α-amylase inhibitors (AAIs) are knottin-type proteins with selective inhibitory potential against coleopteran α-amylases. Their small size and remarkable stability make them exciting molecules for protein engineering to achieve superior selectivity and efficacy. In this report, we have designed a set of AAI pro- and mature peptides chimeras. Based on in silico analysis, stable AAI chimeras having a stronger affinity with target amylases were selected for characterization. In vitro studies validated that chimera of the propeptide from <i>Chenopodium quinoa</i> α-AI and mature peptide from <i>Beta vulgaris</i> α-AI possess 3, 7.6, and 4.26 fold higher inhibition potential than parental counterparts. Importantly, recombinant AAI chimera retained specificity towards target coleopteran α-amylases. In addition, to improve the inhibitory potential of AAI, we performed in silico site-saturation mutagenesis. Computational analysis followed by experimental data showed that substituting Asparagine at the 6th position with Methionine had a remarkable increase in the specific inhibition potential of <i>Amaranthus hypochondriacus</i> α-AI. These results provide structural–functional insights into the vitality of AAI propeptide and a potential hotspot for mutagenesis to enhance the AAI activity. Our investigation will be a toolkit for AAI’s optimization and functional differentiation for future biotechnological applications.</p></div>","PeriodicalId":793,"journal":{"name":"The Protein Journal","volume":"42 5","pages":"519 - 532"},"PeriodicalIF":1.9000,"publicationDate":"2023-08-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"The Protein Journal","FirstCategoryId":"2","ListUrlMain":"https://link.springer.com/article/10.1007/s10930-023-10148-y","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0

Abstract

Amaranthaceae α-amylase inhibitors (AAIs) are knottin-type proteins with selective inhibitory potential against coleopteran α-amylases. Their small size and remarkable stability make them exciting molecules for protein engineering to achieve superior selectivity and efficacy. In this report, we have designed a set of AAI pro- and mature peptides chimeras. Based on in silico analysis, stable AAI chimeras having a stronger affinity with target amylases were selected for characterization. In vitro studies validated that chimera of the propeptide from Chenopodium quinoa α-AI and mature peptide from Beta vulgaris α-AI possess 3, 7.6, and 4.26 fold higher inhibition potential than parental counterparts. Importantly, recombinant AAI chimera retained specificity towards target coleopteran α-amylases. In addition, to improve the inhibitory potential of AAI, we performed in silico site-saturation mutagenesis. Computational analysis followed by experimental data showed that substituting Asparagine at the 6th position with Methionine had a remarkable increase in the specific inhibition potential of Amaranthus hypochondriacus α-AI. These results provide structural–functional insights into the vitality of AAI propeptide and a potential hotspot for mutagenesis to enhance the AAI activity. Our investigation will be a toolkit for AAI’s optimization and functional differentiation for future biotechnological applications.

Abstract Image

查看原文
分享 分享
微信好友 朋友圈 QQ好友 复制链接
本刊更多论文
区域洗牌和位点饱和诱变增强苋科α-淀粉酶抑制剂的抑制潜力
苋科α-淀粉酶抑制剂(AAIs)是一种对鞘翅目α-淀粉酶具有选择性抑制潜力的结蛋白型蛋白。它们的小尺寸和显著的稳定性使它们成为蛋白质工程中令人兴奋的分子,以实现优越的选择性和功效。在本报告中,我们设计了一套AAI前肽和成熟肽嵌合体。基于硅分析,选择与目标淀粉酶亲和力较强的稳定AAI嵌合体进行表征。体外实验证实,藜麦前肽α-AI与甜菜成熟肽α-AI嵌合体的抑制潜能分别比亲本高3倍、7.6倍和4.26倍。重要的是,重组AAI嵌合体保留了对目标鞘翅目α-淀粉酶的特异性。此外,为了提高AAI的抑制潜能,我们进行了硅位点饱和诱变。计算分析和实验数据表明,用蛋氨酸取代第6位的天冬酰胺可显著提高苋α-AI的特异性抑制电位。这些结果为了解AAI前肽的活性提供了结构-功能方面的见解,并为增强AAI活性提供了潜在的诱变热点。我们的研究将成为未来生物技术应用中AAI优化和功能分化的工具包。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 去求助
来源期刊
The Protein Journal
The Protein Journal 生物-生化与分子生物学
CiteScore
5.20
自引率
0.00%
发文量
57
审稿时长
12 months
期刊介绍: The Protein Journal (formerly the Journal of Protein Chemistry) publishes original research work on all aspects of proteins and peptides. These include studies concerned with covalent or three-dimensional structure determination (X-ray, NMR, cryoEM, EPR/ESR, optical methods, etc.), computational aspects of protein structure and function, protein folding and misfolding, assembly, genetics, evolution, proteomics, molecular biology, protein engineering, protein nanotechnology, protein purification and analysis and peptide synthesis, as well as the elucidation and interpretation of the molecular bases of biological activities of proteins and peptides. We accept original research papers, reviews, mini-reviews, hypotheses, opinion papers, and letters to the editor.
期刊最新文献
Influence of Cataract Causing Mutations on αA-Crystallin: A Computational Approach Unraveling the interaction between a glycolytic regulator protein EhPpdk and an anaphase promoting complex protein EhApc10: yeast two hybrid screening, in vitro binding assays and molecular simulation study Unravelling the Significance of Seed Proteomics: Insights into Seed Development, Function, and Agricultural Applications HaloClass: Salt-Tolerant Protein Classification with Protein Language Models Exosomes with Engineered Brain Derived Neurotrophic Factor on Their Surfaces Can Proliferate Menstrual Blood Derived Mesenchymal Stem Cells: Targeted Delivery for a Protein Drug
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
现在去查看 取消
×
提示
确定
0
微信
客服QQ
Book学术公众号 扫码关注我们
反馈
×
意见反馈
请填写您的意见或建议
请填写您的手机或邮箱
已复制链接
已复制链接
快去分享给好友吧!
我知道了
×
扫码分享
扫码分享
Book学术官方微信
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术
文献互助 智能选刊 最新文献 互助须知 联系我们:info@booksci.cn
Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。
Copyright © 2023 Book学术 All rights reserved.
ghs 京公网安备 11010802042870号 京ICP备2023020795号-1