Kinetics of Hydrogen/Deuterium Exchange of EGFR Ligands

¹ University of Puerto Rico, Mayaguez Campus
² Universiry of Puerto Rico, Mayaguez Campus
³ Universiry of Puerto Rico Medical Sciences Campus
⁴ Universiry of Puerto Rico

^* To whom correspondence should be addressed. E-mail: belinda.pastrana{at}gmail.com.

Submitted on November 30, 2007
Revised on December 21, 2007
Accepted on 21 December 2007

	Abstract

Five highly homologous epidermal growth factor receptor (EGFR) ligands were studied by mass spectral analysis, Hydrogen/Deuterium (H/D) exchange via attenuated total reflectance FT-IR spectroscopy, and two-dimensional correlation (2DCOS) 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), heparin binding-EGF (HB-EGF) was determined. All ligands were found to have similar contributions of 3₁₀-helix and random coil with varying contributions of -sheets and -turns. The extent of exchange was 40%, 65%, 55%, 65%, and 98% for EGF, 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, -turns), and 1.88 x 10^-3 min^-1 (-sheets), while for TGF- 0.91 min^-1 (Tyr), 0.27 min^-1 (Arg, -turns), and 1.41 x 10^-4 min^-1 (-sheets). The time constants for AR 0.47 min^-1 (Tyr), 0.04 min^-1 (Arg), and 1.00x10^-4 min^-1 (buried 3₁₀-helix, -turns, and -sheets), for HB-EGF 0.89 min^-1 (Tyr), 0.14 min^-1 (Arg and 3₁₀-helix), and 1.00x10^-3 min^-1 (buried 3₁₀-helix, -sheets, and -turns), and for ER 0.16 min^-1 (Tyr), 0.03 min^-1 (Arg), and 1.00x10^-4 min^-1 3₁₀-helix and -sheets). These results provide essential information towards the understanding solvation, H/D exchange kinetics and conformation of these EGFR ligands in there unbound state.

Key Words: 2DCOS, EGFR ligands, FT-IR spectroscopy, H/D exchange kinetics, MS analysis, secondary structure