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

Abstract

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.

Figure 1.

(A) Labeling scheme of Chymotrypsin inhibitor 2 (CI2), represented in surface-accessible area mode (gray). The unique Cys40 is indicated by a solid black dot and labeled (C40). The added flexible N-terminal TGase tag is depicted by a solid black line with the reactive Gln indicated by a solid black dot and labeled (Q4′). A488-maleimide (A488) and A647-cadaverine (A647) are shown as open circles. (B) Location and solvent accessibility of the internal Glns (dark gray) in CI2 (light gray). (C) Labeling specificity of TGase. Absorbance spectra obtained with CI2-(A488)C40 (solid line) or TG-CI2-(A488)C40 (dotted line) after incubation with a 10-fold stoichiometric excess of A647 in the presence of TGase. Unreacted A647 was removed by filtration prior to acquisition of spectra. For comparison, spectra are normalized to the absorbance at 488 nm.

Figure 2.

Single-molecule FRET. (A) Schematic 2D E–S histogram showing the expected location of A488-only CI2, A647-only CI2, and A647/A488-labeled CI2 (folded protein, high-E; denatured protein, lower E). (B–D) Measured E–S histogram for A647/A488-labeled CI2 at 2 M GdnCl (B), 3.7 M GdnCl (midpoint of folding) (C), and 5 M GdnCl (D). The area of the E–S histograms selected for projection of the 1D E histograms (shown in E) is indicated by a dashed black box. (E) 1D E histograms of A647/A488-labeled CI2 at various concentrations of GdnCl. The solid red lines are fits of the data to Gaussian functions.

Figure 3.

Equilibrium denaturation curves of (A) CI2-C40, (B) TG-CI2-C40, and (C) (A647) TG-CI2-(A488)C40 (measured by ensemble FRET). (D) Normalized curves are shown in A–C (same symbols used). The black-filled dots are data obtained from fitting the single-molecule FRET histograms shown in Figure 2E.