{"title":"Single-molecule tethering methods for membrane proteins.","authors":"Daehyo Lee, Duyoung Min","doi":"10.1016/bs.mie.2023.12.013","DOIUrl":null,"url":null,"abstract":"<p><p>Molecular tethering of a single membrane protein between the glass surface and a magnetic bead is essential for studying the structural dynamics of membrane proteins using magnetic tweezers. However, the force-induced bond breakage of the widely-used digoxigenin-antidigoxigenin tether complex has imposed limitations on its stable observation. In this chapter, we describe the procedures of constructing highly stable single-molecule tethering methods for membrane proteins. These methods are established using dibenzocyclooctyne click chemistry, traptavidin-biotin binding, SpyCatcher-SpyTag conjugation, and SnoopCatcher-SnoopTag conjugation. The molecular tethering approaches allow for more stable observation of structural transitions in membrane proteins under force.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Methods in enzymology","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1016/bs.mie.2023.12.013","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/1/15 0:00:00","PubModel":"Epub","JCR":"Q3","JCRName":"Biochemistry, Genetics and Molecular Biology","Score":null,"Total":0}
引用次数: 0
Abstract
Molecular tethering of a single membrane protein between the glass surface and a magnetic bead is essential for studying the structural dynamics of membrane proteins using magnetic tweezers. However, the force-induced bond breakage of the widely-used digoxigenin-antidigoxigenin tether complex has imposed limitations on its stable observation. In this chapter, we describe the procedures of constructing highly stable single-molecule tethering methods for membrane proteins. These methods are established using dibenzocyclooctyne click chemistry, traptavidin-biotin binding, SpyCatcher-SpyTag conjugation, and SnoopCatcher-SnoopTag conjugation. The molecular tethering approaches allow for more stable observation of structural transitions in membrane proteins under force.
期刊介绍：
The critically acclaimed laboratory standard for almost 50 years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Each volume is eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with over 500 volumes the series contains much material still relevant today and is truly an essential publication for researchers in all fields of life sciences, including microbiology, biochemistry, cancer research and genetics-just to name a few. Five of the 2013 Nobel Laureates have edited or contributed to volumes of MIE.
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