Figure 1.
(A) p-ethynylphenylalanine, (B) Cu(I)-chelating ligands, and (C) azide-functionalized reagents.
Figure 2.
Locations of selected residues for pEthF incorporation in the three-dimensional structure of the mDHFR-WT.
Valine at the 43rd position and phenylalanine at the 179th position are highlighted in red (left) and blue (right), respectively. The cofactor NADPH (magenta) and the inhibitor (orange) are shown in stick representation (PDB ID: 3D80).
Figure 3.
Incorporation of pEthF into the 43rd position of the mDHFR and its orthogonal reactivity.
(A) MALDI-TOF spectra of Lys-C-digested fragments derived from the mDHFR-WT (top) and the mDHFR-pEthF (bottom). (B) SDS-PAGE of the mDHFR-pEthF (pEthF) and the mDHFR-WT (WT) reacted with sulforhodamine-azide. The reaction was performed at 25°C by mixing the protein (30 µM) with the dye (60 µM), CuSO4 (1 mM), TBTA (1 mM), and ascorbate (2 mM) in 20 mM phosphate (pH 8.0) plus 30% (v/v) DMSO. The gel was illuminated by the excitation light at 550 nm (fluorescence panel), and then stained with Coomassie brilliant blue (Coomassie panel).
Figure 4.
Performance of TBTA-assisted CuAAC reactions under various DMSO concentrations and their effects on the enzymatic activity.
(A) Kinetic traces detected at various DMSO volume concentrations in the presence of TBTA or in a DMSO-free buffer without TBTA. Reactions were initiated at 25°C by adding ascorbate (2 mM) to a phosphate-buffered (pH 8.0) mixture containing 30 µM of the mDHFR-43pEthF, 60 µM of azidocoumarin, 1 mM of CuSO4, 1 mM TBTA, and appropriate DMSO contents. Fluorescence evolution was recorded at λex = 400 nm and λem = 470 nm. (B) In-gel fluorescence of an equal amount of reaction products. Relative intensities were quantified by densitometry. (C) Effect of DMSO incubation on activity of the mDHFR-43pEthF. After incubation of 30 µM protein in 10 µl of DMSO-free (closed circle) or 30% (v/v) DMSO-containing phosphate buffer (pH 8.0) (open circle) for 15 min at 25°C, the mixture was diluted 36-fold with the assay buffer for activity assay as described in Materials and Methods. Relative activity was calculated based on changes in absorbance for 10 min after initiation of the enzymatic reaction. Error bars represent standard errors (n = 3).
Figure 5.
THPTA-assisted CuAAC reaction and its effect on the enzymatic activity.
(A) In-gel fluorescence of reaction products from TBTA- and THPTA-assisted dye labeling via CuAAC. The mDHFR-pEthF (30 µM) was reacted at 25°C for 15 min with sulforhodamine-azide (60 µM) in the presence of 1 mM CuSO4, 1 mM TBTA, 2 mM ascorbate in a phosphate buffer (pH 8.0) containing 30% (v/v) DMSO or in the presence of 1 mM CuSO4, 1 mM THPTA, 2 mM ascorbate in a DMSO-free phosphate buffer (pH 8.0). (B) Effect of THPTA incubation on activity of the mDHFR-43pEthF. After incubation of 30 µM protein with (open circle) or without (close circle) 1 mM THPTA in 10 µl of DMSO-free phosphate buffer (pH 8.0) for 15 min at 25°C, the mixture was diluted 36-fold with the assay buffer for activity assay. Error bars represent standard errors (n = 3). (C) Effect of THPTA incubation on activity of the mDHFR-179pEthF (n = 3).
Figure 6.
Effect of various reductants on CuAAC reaction rates and activities of the mDHFR-43pEthF and the mDHFR-179pEthF.
(A) Time course of CuAAC reactions initiated by ascorbate, TCEP, and DTT. Reactions were performed at 25°C by adding 2 mM reductant to a phosphate-buffered (pH 8.0) mixture containing 30 µM of the mDHFR-43pEthF (black) or the mDHFR-179pEthF (gray), 60 µM of azidocoumarin, 1 mM of CuSO4, 1 mM THPTA. Fluorescence evolution was recorded at λex = 400 nm and λem = 470 nm. (B) Relative loss of enzymatic activity after incubation with various CuAAC systems in the absence of azidocoumarin (1–4). Incubation times were 15 min for system 1 and 4, 12 h for system 2, and 2 h for system 3. Activity losses were normalized to that in system 1. Error bars represent standard errors (n = 3). Two-sided Student's t-tests were applied to the data (*P<0.05). (C) Effect of ascorbate-driven dye labeling of the mDHFR-43pEthF on the enzymatic activity. The labeling was performed at 25°C for 15 min in the presence of 30 µM of the mDHFR-43pEthF, 60 µM of azidocoumarin, 1 mM of CuSO4, 1 mM THPTA, and 2 mM ascorbate. Error bars represent standard errors (n = 3).
Figure 7.
Enzymatic activity of the mDHFR after site-specific biotinylation and immobilization.
(A) Activity of the mDHFR-43biotin versus the mDHFR-43pEthF. Error bars represent standard errors (n = 3). (B) Activity of the immobilized mDHFR-43biotin. Streptavidin-coated wells were incubated with 50 µL of the mDHFR-43biotin and the mDHFR-43pEthF at 1 mg/mL, separately, for 30 min at RT. After washing, the enzymatic reaction was initiated at 25°C by adding 200 µL of assay buffer, and monitored by spectrometry. Error bars represent standard errors (n = 3).