Bio‐Orthogonal Protein Labeling Methods for Single Molecule FRET

Single‐molecule fluorescence resonance energy transfer (smFRET) has emerged as a powerful technique for visualizing the dynamics and architecture of proteins, multi‐subunit protein complexes, or protein‐nucleic acid complexes. To suffice a FRET study, selective labeling with a pair of a donor dye and an acceptor dye is required, with the two dyes on two sites on a protein, or with the donor on one protein sub‐unit while the acceptor on another subunit, or with the pair divided between protein and DNA (RNA). Due to the availability of orthogonal chemical moieties, most biophysicists armed with single FRET have been limited to the processes of nucleic acids metabolism by studying protein‐DNA (‐RNA) interactions. Nevertheless, diverse biological processes usually involve communications between proteins. Indeed, protein‐protein interactions constitute fundamental questions regarding intra‐ and intercellular signal transduction and are under intensive research, yet mainly by conventional methodologies. In this review, various bio‐orthogonal methods recently developed for labeling proteins via protein domains, small peptides, or single amino acids are examined. These approaches comprise a repertoire that overcomes the challenges of labeling two sites on a protein or a protein complex to facilitate the investigation of protein‐protein interactions by the modern single molecule FRET method.