Kinetic and Structural Characterization of the Self-Labeling Protein Tags HaloTag7, SNAP-tag, and CLIP-tag

Abstract

The self-labeling protein tags (SLPs) HaloTag7, SNAP-tag, and CLIP-tag allow the covalent labeling of fusion proteins with synthetic molecules for applications in bioimaging and biotechnology. To guide the selection of an SLP-substrate pair and provide guidelines for the design of substrates, we report a systematic and comparative study of the labeling kinetics and substrate specificities of HaloTag7, SNAP-tag, and CLIP-tag. HaloTag7 reaches almost diffusion-limited labeling rate constants with certain rhodamine substrates, which are more than 2 orders of magnitude higher than those of SNAP-tag for the corresponding substrates. SNAP-tag labeling rate constants, however, are less affected by the structure of the label than those of HaloTag7, which vary over 6 orders of magnitude for commonly employed substrates. Determining the crystal structures of HaloTag7 and SNAP-tag labeled with fluorescent substrates allowed us to rationalize their substrate preferences. We also demonstrate how these insights can be exploited to design substrates with improved labeling kinetics.

Figure 1

Self-labeling reaction, substrates, and kinetic models. (A) Scheme of the HT7 labeling reaction with fluorophore substrates. The chemical structure of HT7 substrates (CA) is depicted below. R represents the functional moiety to be linked to HT7. (B) Scheme of the SNAP(f)/CLIP(f) labeling reaction with fluorophore substrates. The chemical structures of SNAP/CLIP substrates (BG/CP/BC) are depicted below. R represents the functional moiety to be linked to the SLP. (C) Models employed to describe the SLP kinetics in this study. (D) Popular SLP labels used in this study. Abbreviations: TMR, tetramethylrhodamine; JF, Janelia Fluor dyes; CPY, carbopyronine; BCN, biscyclononyne; SCO, cyclooctyne; Tz, tetrazine; PhN3, phenylazide; Vbn, vinylbenzene; Nor1, (1R,4R)-bicyclo[2.2.1]hept-2-ene; Nor2, (1S,4S)-5-methylbicyclo[2.2.1]hept-2-ene; N3, methylazide; Ac, acetate.

Figure 2

Characterization of HaloTag7 labeling kinetics. (A) Fluorescence anisotropy traces (points) and fitted curves of HT7 labeling with CA-TMR in a 1:1 stoichiometry at the indicated concentrations. Kinetics were recorded by following the fluorescence anisotropy over time using a stopped-flow device. Reactions were started by mixing equal volumes of HT7 and CA-TMR. Data were fitted to kinetic model 2 (lines). (B) HT7 affinities (K_d) for different fluorophore substrates calculated from the kinetic parameters (k_–1 and k₁). (C) HT7 reactivity (k₂) for different fluorophore substrates obtained from fluorescence anisotropy kinetics. The minimal differences in k₂ illustrate that labeling kinetics are mostly influenced by differences in K_d. (D) Apparent second-order labeling rate constants (k_app) of HT7 with different substrates. Rate constants span >6 orders of magnitude. Non-negatively charged fluorophore substrates reach the fastest labeling kinetics. (E) Comparison of k_app between HT7 and HOB for CA-TMR and CA-Alexa488 labeling highlighting the preference of HOB for the negatively charged substrate CA-Alexa488. (F) Correlation between the HT7 apparent second-order rate constant (k_app) and affinity (K_a = 1/K_d) for different fluorophore substrates. Affinities were obtained with the inactive variant HT7^D106A. Log-transformed values were fitted to a linear model [black line, log(k_app) = log(K_a) × 1.042 + 1.544]. The gray area represents the 95% confidence bands (the area in which the true regression line lies with 95% confidence).

Figure 3

Structure–function analysis of HaloTag7–substrate interactions. (A) Structural comparison between HT7-TMR (PDB entry 6Y7A, gray) and HT7-CPY (PDB entry 6Y7B, chain A, gold). Close-ups of the substrate binding sites of both proteins are included. Proteins are represented as gray cartoons, and the fluorophore substrates and residues as sticks. Putative hydrogen bonds are represented as dashed lines with annotated distances. A comparison of the TMR and CPY conformations on HT7 is shown (bottom left). (B) HT7 affinities (K_d) and free binding energies (ΔG) for different TMR substrate substructures. (C) Comparison of HT7 affinity for CA-6-TMR and CA-5-TMR. (D and E) Surface electrostatic potentials of HT7-TMR (PDB entry 6Y7A) and HOB-TMR (PDB entry 6ZCC), respectively. Electrostatic potentials are drawn as protein surfaces from −2.0 (red) to 2.0 (blue) kJ mol^–1 e^–1 and were obtained using the APBS software with standard parameters.

Figure 4

Characterization of SNAP- and CLIP-tag labeling kinetics. (A) Comparison of labeling kinetics (k_app) between SNAP and SNAPf. (B) Correlation between the SNAP apparent second-order rate constant (k_app) and affinity (K_a = 1/K_d) for different fluorophore substrates. Affinities were obtained for inactive variant SNAP^C145A. Log-transformed values were fitted to a linear model [black line, log(k_app) = log(K_a) × 1.0217 – 0.7407]. The gray area represents the 95% confidence bands (the area in which the true regression line lies with 95% confidence). (C) Comparison of labeling kinetics (k_app) between CLIP and CLIPf. (D) Comparison of labeling kinetics (k_app) between SNAPf and CLIPf. (E) Apparent second-order labeling rate constants (k_app) of SNAP with different substrates. Kinetics span >3 orders of magnitude (2 orders of magnitude within each substrate class BG/CP). BG-based, non-negatively charged fluorophore substrates reach the fastest labeling kinetics.

Figure 5

Structure–function analysis of SNAP-tag fluorophore–substrate interactions. (A) Structural comparison between SNAP-TMR (PDB entry 6Y8P) and the BG-bound variant of SNAP^C145A (PDB entry 3KZZ). SNAP is represented as a cartoon, and the ligands and residues are represented as sticks. Putative hydrogen bonds and corresponding distances are indicated by dashed lines. (B) Increase in affinity between SNAP^C145A and SNAPf^C145A for different fluorophore substrates. The number in parentheses indicate different linkages of the fluorophore benzyl group to BG.

Figure 6

Comparison of labeling kinetics between SNAP-tag and HaloTag7. Apparent labeling rate constants (k_app) of HT7 span >6 orders of magnitude, while rate constants of SNAP span only >2 orders of magnitude (BG-substrates). The blue area highlights the span of SNAP apparent labeling rate constants. Depending on the application, some substrates should preferentially be employed with HT7 or SNAP to ensure quick labeling. A rate constant of 10⁵ M^–1 s^–1 corresponds to a half-labeling time of ∼7 s at 1 μM substrate, in excess.