title: Simple and efficient strategy for site-specific dual labeling of proteins for single-molecule fluorescence resonance energy transfer analysis.
abstract: Analysis of protein dynamics using single-molecule fluorescence resonance energy transfer (smFRET) is widely used to understand the structure and function of proteins. Nonetheless, site-specific labeling of proteins with a pair of donor and acceptor dyes still remains a challenge. Here we present a general and facile method for site-specific dual labeling of proteins by incorporating two different, readily available, unnatural amino acids (p-acetylphenylalanine and alkynyllysine) for smFRET. We used newly evolved alkynyllysine-specific aminoacyl-tRNA synthetase/tRNA(UCA) and p-acetylphenylalanyl-tRNA synthetase/tRNA(CUA). The utility of our approach was demonstrated by analyzing the conformational change of dual-labeled calmodulin using smFRET measurements. The present labeling approach is devoid of major limitations in conventional cysteine-based labeling. Therefore, our method will significantly increase the repertoire of proteins available for FRET study and expand our ability to explore more complicated molecular dynamics.
authors.affiliation: Department of Chemistry, Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Korea.
authors.forename: Jihyo
authors.initials: J
authors.lastname: Kim
authors.forename: Moon-Hyeong
authors.initials: MH
authors.lastname: Seo
authors.forename: Sangsik
authors.initials: S
authors.lastname: Lee
authors.forename: Kyukwang
authors.initials: K
authors.lastname: Cho
authors.forename: Aerin
authors.initials: A
authors.lastname: Yang
authors.forename: Kyunghwa
authors.initials: K
authors.lastname: Woo
authors.forename: Hak-Sung
authors.initials: HS
authors.lastname: Kim
authors.forename: Hee-Sung
authors.initials: HS
authors.lastname: Park
mesh_terms: D001483:Base Sequence; D001665:Binding Sites / Q000502:physiology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012343:RNA, Transfer / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D011506:Proteins; D012343:RNA, Transfer