PMC

Mass spectrometric product analysis of TG2-catalyzed cross-linking reactions between the SnQ1 peptide and amine-donor compounds. MALDI/MS spectrum of aliquots from reactions between SnQ1 and (A) 5-(biotinamido)penthylamine (5BP) or (B) biotinyl-GPAVTAAPKK (BKP) after incubation in the presence of TG2. Signal assignments are given in .

Relative occurrence of amino acids in the environment of glutamines within the selected glutamine-containing peptides compared to those of the initial library. Amino acid frequencies are shown for relative positions Q − 1, Q + 1, Q + 2, and Q + 3 (top diagrams), and the values for the initial library are presented in the bottom diagram.

Tandem mass spectrometric identification of TG2-modified glutamine residues in SnQ1. An aliquot of the TG2 reaction mixture of SnQ1 and BKP was resolved using μLC-ESI/MS, and the triply charged ion at m/z 1414.98, originating from the SnQ1 peptide cross-linked to biotinyl-GPAVTAAPKK (BKP), was selected for fragmentation. The MS/MS spectrum obtained from the fragmentation of the above precursor ion is shown below. Peptide sequences and interpretation are given above. Signals X1–X6 are assigned to cross-linked fragments, which are structurally explained on the right. Dashed lines depict the three alternative ɛ(γ-glutamyl)lysine isopeptide bonds between the two peptides as deduced from fragments X1–X6.

Circular dichroism–based analysis of the structure of the SnQ1 peptide. (A) Far-UV CD spectra of SnQ1 (1 mg/mL) recorded in a 0.1-mm cuvette (Hellma), with 8-sec response time, in 10 mM phosphate buffer (pH 7.0) in the absence (black line) and the presence of 10% (black dashed line), 20% (light gray), and 50% (dark gray) trifluoroethanol. The inset shows a plot of ellipticity at 195 nm (open circle), 222 nm (closed circle), and 228 nm (open square) against the TFE concentration. (B) Near-UV CD spectra of the peptide (0.1 mg/mL, 1-mm cuvette) in the absence (continuous line) and the presence of 50% TFE (dashed line).

Kinetics of binding and transamidation of the SnQ1 peptide by TG2. (A) For surface plasmon resonance spectroscopic analysis of binding, a streptavidin-coated (SA) chip was charged with biotinyl-SnQ1 (2 μg/mL) to reach a 190, 630, and 900 response unit increase in channels 1, 2, and 3, respectively. Five microliters of GST-TG2 (2 μg/mL) was passed over the chip surface in running buffer (10 mM Tris-HCl at pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.01% P20). The profiles for channels 1, 2, and 3 are shown after subtraction of the control. (B) Analysis of the progress of ammonia release when TG2 is incubated with 50 μM (open circles), 100 μM (closed circles), 200 μM (open squares), and 400 μM (closed squares) SnQ1 in the presence of 80 mM ethylamine as the amine-donor substrate and 5 mM CaCl₂, using a continuous spectrophotometric linked enzyme assay as described in Materials and Methods. (C) Determination of kinetic parameters (V_max and K_M) of SnQ1 transamidation by nonlinear fitting of the Michaelis-Menten equation onto the initial velocities calculated by linear fitting of the corresponding progress curves and plotted against SnQ1 concentration.

TG2-catalyzed modification of phage-displayed peptides from selection rounds 2 and 3. Phage particles were incubated at 37°C for 30 min in the presence of 5 mM CaCl₂, 2 mM 5-(biotinamido)penthylamine (5BP), and 2 μg of GST-transglutaminase 2 (GST-TG2). Phage proteins were resolved on 10% SDS-PAGE and analyzed by Western blotting. Biotinylated protein bands were detected by streptavidin-biotinylated horseradish peroxidase conjugate and visualized by a chemiluminescent technique. (Lane 1) GST-TG2 incubated with 5BP; (lane 2) GST-TG2 incubated with 5BP and 5 mM EDTA as a negative control; (lanes 3–13) phage clones incubated with GST-TG2 and 5BP; (lane 3) PhQ2.7 (GQQQTPY); (lane 4) PhQ2.8 (GLQATPA); (lane 5) PhQ2.10 (VQETQRP); (lane 6) PhQ2.2 (GLQQASV); (lane 7) PhQ2.5 (QGRIPTS); (lane 8) phage clone displaying the peptide MPPPMRS as non-Q control; (lane 9) PhQ2.4 (VTQRLPL); (lane 10) PhQ3.11 (WQTPMNS); (lane 11) PhQ3.13 (QQLHLEA); (lane 12) phage clone displaying the peptide LMAKPTR as non-Q control; (lane 13) PhQ3.19 (SQLTLLP). Closed arrowheads mark bands corresponding to GST-TG2 species with incorporated 5BP. The open arrowhead marks the position of the gene III protein of M13 phage, which runs at an apparent molecular mass of 62 kDa. Lanes 1–9 and lanes 10–13 are from separate experiments.