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The kinetics of the hydrogen/deuterium exchange of epidermal growth factor receptor ligands.

Iloro I, Narváez D, Guillén N, Camacho CM, Guillén L, Cora E, Pastrana-Ríos B - Biophys. J. (2008)

Bottom Line: All ligands were found to have similar contributions of 3(10)-helix and random coil with varying contributions of beta-sheets and beta-turns.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, beta-turns, and beta-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, beta-sheets, and beta-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 beta-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.

View Article: PubMed Central - PubMed

Affiliation: Center for Protein Structure Function and Dynamics, University of Puerto Rico, Mayagüez Campus, Mayagüez, Puerto Rico.

ABSTRACT
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 beta-sheets and beta-turns. The extent of exchange was 40%, 65%, 55%, 65%, and 98% for EGF, transforming growth factor-alpha (TGF-alpha), 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, beta-turns), and 1.88 x 10(-3) min(-1) (beta-sheets and 3(10)-helix); and for TGF-alpha 0.91 min(-1) (Tyr), 0.27 min(-1) (Arg, beta-turns), and 1.41 x 10(-4) min(-1) (beta-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, beta-turns, and beta-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, beta-sheets, and beta-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 beta-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.

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Overlaid spectra of EGFR ligands corresponding to initial and final spectra collected during the H/D exchange experiment within the spectral region of 3600–1400 cm−1 for (A) EGF, (B) TGF-α, (C) AR, (D) HB-EGF, and (E) ER. The spectra demonstrate the extent of hydration of the protein film and the overall band shifts and intensity changes for all five ligands.
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fig3: Overlaid spectra of EGFR ligands corresponding to initial and final spectra collected during the H/D exchange experiment within the spectral region of 3600–1400 cm−1 for (A) EGF, (B) TGF-α, (C) AR, (D) HB-EGF, and (E) ER. The spectra demonstrate the extent of hydration of the protein film and the overall band shifts and intensity changes for all five ligands.

Mentions: Typical ATR-FT-IR spectra for a D2O hydrated uniform protein film in the spectral region of 3600–1400 cm−1 for EGF, TGF-α, AR, HB-EGF, and ER (Fig. 3, A–E, respectively) are composed of the amide A band (∼3200 cm−1), the amide I′ band (∼1650 cm−1), the amide II band (∼1540 cm−1), and the amide II′ band (∼1450 cm−1). We closely monitored the spectral changes in the spectral region 1700–1400 cm−1 during H→D exchange to include the observed shift of the amide I′ band form accompanied by an increase in bandwidth. In addition, a decrease in intensity of the amide II band at ∼1540 cm−1 along with a concomitant increase of the amide II′ band at ∼1450 cm−1 were observed. These spectral changes were studied in detail by curve fitting analysis, difference spectroscopy, 2DCOS analysis, and H/D exchange kinetics.


The kinetics of the hydrogen/deuterium exchange of epidermal growth factor receptor ligands.

Iloro I, Narváez D, Guillén N, Camacho CM, Guillén L, Cora E, Pastrana-Ríos B - Biophys. J. (2008)

Overlaid spectra of EGFR ligands corresponding to initial and final spectra collected during the H/D exchange experiment within the spectral region of 3600–1400 cm−1 for (A) EGF, (B) TGF-α, (C) AR, (D) HB-EGF, and (E) ER. The spectra demonstrate the extent of hydration of the protein film and the overall band shifts and intensity changes for all five ligands.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2367206&req=5

fig3: Overlaid spectra of EGFR ligands corresponding to initial and final spectra collected during the H/D exchange experiment within the spectral region of 3600–1400 cm−1 for (A) EGF, (B) TGF-α, (C) AR, (D) HB-EGF, and (E) ER. The spectra demonstrate the extent of hydration of the protein film and the overall band shifts and intensity changes for all five ligands.
Mentions: Typical ATR-FT-IR spectra for a D2O hydrated uniform protein film in the spectral region of 3600–1400 cm−1 for EGF, TGF-α, AR, HB-EGF, and ER (Fig. 3, A–E, respectively) are composed of the amide A band (∼3200 cm−1), the amide I′ band (∼1650 cm−1), the amide II band (∼1540 cm−1), and the amide II′ band (∼1450 cm−1). We closely monitored the spectral changes in the spectral region 1700–1400 cm−1 during H→D exchange to include the observed shift of the amide I′ band form accompanied by an increase in bandwidth. In addition, a decrease in intensity of the amide II band at ∼1540 cm−1 along with a concomitant increase of the amide II′ band at ∼1450 cm−1 were observed. These spectral changes were studied in detail by curve fitting analysis, difference spectroscopy, 2DCOS analysis, and H/D exchange kinetics.

Bottom Line: All ligands were found to have similar contributions of 3(10)-helix and random coil with varying contributions of beta-sheets and beta-turns.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, beta-turns, and beta-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, beta-sheets, and beta-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 beta-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.

View Article: PubMed Central - PubMed

Affiliation: Center for Protein Structure Function and Dynamics, University of Puerto Rico, Mayagüez Campus, Mayagüez, Puerto Rico.

ABSTRACT
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 beta-sheets and beta-turns. The extent of exchange was 40%, 65%, 55%, 65%, and 98% for EGF, transforming growth factor-alpha (TGF-alpha), 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, beta-turns), and 1.88 x 10(-3) min(-1) (beta-sheets and 3(10)-helix); and for TGF-alpha 0.91 min(-1) (Tyr), 0.27 min(-1) (Arg, beta-turns), and 1.41 x 10(-4) min(-1) (beta-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, beta-turns, and beta-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, beta-sheets, and beta-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 beta-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.

Show MeSH
Related in: MedlinePlus