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FOR THE RECORD

Site-specific labeling of proteins for single-molecule FRET by combining chemical and enzymatic modification

¹ Department of Chemistry and Biochemistry, ² Department of Physiology, and ³ Molecular Biology Institute, University of California at Los Angeles (UCLA), Los Angeles, California 90095, USA

(RECEIVED September 16, 2005; FINAL REVISION November 22, 2005; ACCEPTED November 28, 2005)

An often limiting factor for studying protein folding by single-molecule fluorescence resonance energy transfer (FRET) is the ability to site-specifically introduce a photostable organic FRET donor (D) and a complementary acceptor (A) into a polypeptide chain. Using alternating-laser excitation and chymotrypsin inhibitor 2 as a model, we show that chemical labeling of a unique cysteine, followed by enzymatic modification of a reactive glutamine in an N-terminally appended substrate sequence recognition tag for transglutaminase (TGase) affords stoichiometrically D-/A-labeled protein suitable for single-molecule FRET experiments. Thermodynamic data indicate that neither the presence of the TGase tag nor D/A labeling perturbs protein stability. As the N terminus in proteins is typically solvent accessible, a TGase tag can (in principle) be appended to any protein of interest by genetic engineering. Two-step chemical/enzymatic labeling may thus represent a simple, low-cost, and widely available strategy for D/A labeling of proteins for FRET-based single-molecule protein folding studies, even for non-protein-experts laboratories.

Keywords: protein labeling; transglutaminase; fluorescence resonance energy transfer; single-molecule spectroscopy; alternating laser excitation; fluorescence-aided molecular sorting

Abbreviations: A, FRET acceptor • A488, Alexa488 maleimide • A647, Alexa647 cadaverine • ALEX, alternating-laser excitation • CI2, chymotrypsin inhibitor 2 • D, FRET donor • E, FRET efficiency • FAMS, fluorescence-aided molecular sorting • FRET, fluorescence resonance energy transfer • IEX, ion-exchange chromatography • MW, molecular weight • R₀, Förster radius • S, ALEX-ratio S • TGase, transglutaminase

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051851506.

Reprint requests to: Shimon Weiss, Department of Chemistry and Biochemistry, UCLA, 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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