Site-specific labeling of proteins for single-molecule FRET by combining chemical and enzymatic modification.
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.
Jäger
Marcus
M
Department of Chemistry and Biochemistry, UCLA, Charles E. Young Drive East, Los Angeles, CA 90095, USA.
Nir
Eyal
E
Weiss
Shimon
S
D003545:Cysteine / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D005973:Glutamine / Q000737:chemistry; D010455:Peptides / Q000737:chemistry / Q000235:genetics; D010940:Plant Proteins; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013816:Thermodynamics; D011503:Transglutaminases
D005456:Fluorescent Dyes; D010455:Peptides; D010940:Plant Proteins; C093336:chymotrypsin inhibitor 2; D005973:Glutamine; D011503:Transglutaminases; D003545:Cysteine