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

Single-molecule Forster resonance energy transfer (smFRET) is a powerful tool for investigating the dynamic properties of biomacromolecules. However, the success of protein smFRET relies on the precise and efficient labeling of two or more fluorophores on the protein of interest (POI), which has remained highly challenging, particularly for large membrane protein complexes. Here, we demonstrate the site-selective incorporation of a novel unnatural amino acid (2-amino-3-(4-hydroselenophenyl) propanoic acid, SeF) through genetic expansion followed by a Se-click reaction to conjugate the Bodipy593 fluorophore on calmodulin (CaM) and beta-arrestin-1 (beta arr1). Using this strategy, we monitored the subtle but functionally important conformational change of beta arr1 upon activation by the G-protein coupled receptor (GPCR) through smFRET for the first time. Our new method has broad applications for the site-specific labeling and smFRET measurement of membrane protein complexes, and the elucidation of their dynamic properties such as transducer protein selection.

Automated summary

smFRET is a powerful tool for studying the dynamic properties of biomacromolecules, but efficient labeling of multiple fluorophores on proteins remains challenging. This study demonstrates a novel method using a unique unnatural amino acid for precise labeling and smFRET monitoring of protein complexes, allowing the observation of subtle conformational changes of proteins upon activation. This approach has broad implications for understanding the dynamic properties of membrane protein complexes and transducer protein selection.