Single-molecule FRET and conformational analysis of beta-arrestin-1 through genetic code expansion and a Se-click reaction

• DOI: 10.1039/d1sc02653d
• Publication Date: 2021-05-31T08:23:41Z
• Authors: Ming-Jie Han; Qing-tao He; Mengyi Yang; Chao Chen; Yirong Yao; Xiaohong Liu; Yuchuan Wang; Zhong-liang Zhu; Kong-kai Zhu; Changxiu Qu; Fan Yang; Cheng Hu; Xuzhen Guo; Dawei Zhang; Chunlai Chen; Jin-peng Sun; Jiangyun Wang
• Keywords: Single-molecule FRET; Genetic Code

Single-molecule Förster resonance energy transfer (smFRET) is a powerful tool for investigating the dynamic properties of biomacromolecules. However, success protein smFRET relies on precise and efficient labeling two or more fluorophores interest (POI), which has remained highly challenging, particularly large membrane complexes. Here, we demonstrate site-selective incorporation novel unnatural amino acid (2-amino-3-(4-hydroselenophenyl) propanoic acid, SeF) through genetic expansion followed by Se-click reaction to conjugate Bodipy593 fluorophore calmodulin (CaM) β-arrestin-1 (βarr1). Using this strategy, monitored subtle but functionally important conformational change βarr1 upon activation G-protein coupled receptor (GPCR) first time. Our new method broad applications site-specific measurement complexes, elucidation their such as transducer selection.