Protein-protein interactions as a tool for site-specific labeling of proteins.
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.
Jäger
Marcus
M
Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA.
Michalet
Xavier
X
Weiss
Shimon
S
D003545:Cysteine / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D013058:Mass Spectrometry; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry / Q000235:genetics; D010940:Plant Proteins; D017510:Protein Folding; D025941:Protein Interaction Mapping; D011994:Recombinant Proteins / Q000737:chemistry*; D013381:Subtilisins / Q000737:chemistry / Q000235:genetics
D005456:Fluorescent Dyes; D010455:Peptides; D010940:Plant Proteins; D011994:Recombinant Proteins; C093336:chymotrypsin inhibitor 2; D013381:Subtilisins; D003545:Cysteine