The Kinetics of the Hydrogen/Deuterium Exchange of Epidermal Growth Factor Receptor Ligands

Five highly homologous epidermal growth factor receptor ligands were studied by mass spectral analysis, hydrogen/deuterium (H/D) exchange via attenuated total reflectance Fourier transform-infrared spectroscopy, and two-dimensional correlation analysis. These studies were performed to determine the order of events during the exchange process, the extent of H/D exchange, and associated kinetics of exchange for a comparative analysis of these ligands. Furthermore, the secondary structure composition of amphiregulin (AR) and heparin-binding-epidermal growth factor (HB-EGF) was determined. All ligands were found to have similar contributions of 3(10)-helix and random coil with varying contributions of β-sheets and β-turns. The extent of exchange was 40%, 65%, 55%, 65%, and 98% for EGF, transforming growth factor-α (TGF-α), AR, HB-EGF, and epiregulin (ER), respectively. The rate constants were determined and classified as fast, intermediate, and slow: for EGF the 0.20 min(−1) (Tyr), 0.09 min(−1) (Arg, β-turns), and 1.88 × 10(−3) min(−1) (β-sheets and 3(10)-helix); and for TGF-α 0.91 min(−1) (Tyr), 0.27 min(−1) (Arg, β-turns), and 1.41 × 10(−4) min(−1) (β-sheets). The time constants for AR 0.47 min(−1) (Tyr), 0.04 min(−1) (Arg), and 1.00 x 10(−4) min(−1) (buried 3(10)-helix, β-turns, and β-sheets); for HB-EGF 0.89 min(−1) (Tyr), 0.14 min(−1) (Arg and 3(10)-helix), and 1.00 x 10(−3) min(−1) (buried 3(10)-helix, β-sheets, and β-turns); and for epiregulin 0.16 min(−1) (Tyr), 0.03 min(−1) (Arg), and 1.00 x 10(−4) min(−1) (3(10)-helix and β-sheets). These results provide essential information toward understanding secondary structure, H/D exchange kinetics, and solvation of these epidermal growth factor receptor ligands in their unbound state.