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

Ibon Iloro ^*, Daniel Narváez ^*, Nancy Guillén , Carlos M. Camacho , Lalisse Guillén , Elsa Cora ^*  and Belinda Pastrana-Ríos ^* 

^* Center for Protein Structure Function and Dynamics, and Department of Chemistry, University of Puerto Rico, Mayagüez Campus, Mayagüez, Puerto Rico 00681-9019; and Department of Biochemistry, University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico 00936-5067

Correspondence: Address reprint requests to Belinda Pastrana-Ríos, Dept. of Chemistry, University of Puerto Rico, Mayagüez Campus, PO Box 9019, Mayagüez, Puerto Rico 00681-9019. E-mail: belinda{at}hpcf.upr.du.

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 3₁₀-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- (TGF-), 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 x 10^–3 min^–1 (β-sheets and 3₁₀-helix); and for TGF- 0.91 min^–1 (Tyr), 0.27 min^–1 (Arg, β-turns), and 1.41 x 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 3₁₀-helix, β-turns, and β-sheets); for HB-EGF 0.89 min^–1 (Tyr), 0.14 min^–1 (Arg and 3₁₀-helix), and 1.00 x 10^–3 min^–1 (buried 3₁₀-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 (3₁₀-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.