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

¹ Departments of Chemistry and Biochemistry and ² Physiology, University of California at Los Angeles (UCLA), Los Angeles, California 90095, USA

(RECEIVED February 9, 2005; FINAL REVISION February 9, 2005; ACCEPTED April 17, 2005)

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.

Keywords: protein labeling; protein folding; protein–protein interaction; fluorescence resonance energy transfer (FRET); single molecule spectroscopy; alternating laser excitation; fluorescence-aided molecular sorting

Abbreviations: A, FRET-acceptor • A488, Alexa Fluor 488 • A647, Alexa Fluor 647 • ALEX, alternating laser excitation, a novel single-molecule spectroscopic technique • CI2, chymotrypsin inhibitor 2 • Cys, cysteine • D, FRET-donor • DTT, dithiotreitol • E, FRET-efficiency • FAMS, fluorescence-aided molecular sorting • FRET, fluorescence resonance energy transfer • F_U, fraction of unfolded protein • GdnCl, guanidinium chloride • GdnSCN, guanidinium thiocyanate • K_D, dissociation constant • R₀, Förster radius • S, ALEX-ratio S, accounting for D/A-stoichometry in D/A-labeled biomolecules • Sbt BPN', subtilisin BPN', a serine protease • SLOPPI, site-specific labeling of proteins using protein–protein interactions • spFRET, single-pair FRET • wt, wild type.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051384705.

Reprint requests to: Shimon Weiss, Department of Chemistry and Biochemistry, UCLA, 607 Charles E. Young Drive East, Los Angeles, CA 90095, USA; e-mail: sweiss{at}chem.ucla.edu; fax: (310) 267-4672.

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