Protein–protein interactions as a tool for site‐specific labeling of proteins

Abstract

Probing structures and dynamics within biomolecules using ensemble and single‐molecule fluorescence resonance energy transfer requires the conjugation of fluorophores to proteins in a site‐specific and thermodynamically nonperturbative fashion. Using single‐molecule fluorescence‐aided molecular sorting and the chymotrypsin inhibitor 2–subtilisin BPN′ complex as an example, we demonstrate that protein–protein interactions can be exploited to afford site‐specific labeling of a recombinant double‐cysteine variant of CI2 without the need for extensive and time‐consuming chromatography. The use of protein–protein interactions for site‐specific labeling of proteins is compatible with and complementary to existing chemistries for selective labeling of N‐terminal cysteines, and could be extended to label multiple positions within a given polypeptide chain.