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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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The kinetics of exchange for ER is summarized in three plots. (A) Initial times of exchange. (B) Percent of unexchanged protein versus time. ER was observed to exchange 98% after 150 min. A straight line shown at the bottom of each multiexponential decay plot (A and B) is the residual of the fit with the resulting standard deviation 0.650. (C) Evolution of the absorbance at selected wavenumbers versus time, which includes a summary of the kinetic components assigned to Tyr, Arg, and the different structural motifs.
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fig9: The kinetics of exchange for ER is summarized in three plots. (A) Initial times of exchange. (B) Percent of unexchanged protein versus time. ER was observed to exchange 98% after 150 min. A straight line shown at the bottom of each multiexponential decay plot (A and B) is the residual of the fit with the resulting standard deviation 0.650. (C) Evolution of the absorbance at selected wavenumbers versus time, which includes a summary of the kinetic components assigned to Tyr, Arg, and the different structural motifs.

Mentions: The overall extent of exchange for each ligand is shown in Figs. 5, A and B, to 9, A and B, and is summarized in Table 1. In general, the extent of exchange agreed with the overall hydrophobic residue content for each ligand (as analyzed by the Kyte-Doolittle hydropathy sequence analysis (60)), oligomeric state, and glycosylation. The exception was AR, suggesting that the polar residues may be buried, whereas the hydrophobic residues mainly located within C-terminal end are exposed. The fastest initial exchange was observed for TGF-α: within the first 5 min of the exchange process, 50% of the ligand had exchanged. The next fastest initial exchange was HB-EGF: within the first 5 min, 40% had exchanged, probably due to the glycosylation. Although ER had the slowest initial exchange, it was also this ligand that underwent complete exchange, suggesting that the dimer species is present in small quantities and that this protein is almost completely exposed to its aqueous environment. The extent of H/D exchange for these ligands, with EGF having the lowest extent of exchange, suggests the dimer form contributed to the loss of exposure to its aqueous environment with only 40%; in contrast, epiregulin had almost complete exchange (98%). HB-EGF had a greater extent of exchange, i.e., 65%, suggesting the glycosylation affected hydration of the ligand, whereas TGF-α and AR had extents of exchange of 65% and 55%, respectively.


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)

The kinetics of exchange for ER is summarized in three plots. (A) Initial times of exchange. (B) Percent of unexchanged protein versus time. ER was observed to exchange 98% after 150 min. A straight line shown at the bottom of each multiexponential decay plot (A and B) is the residual of the fit with the resulting standard deviation 0.650. (C) Evolution of the absorbance at selected wavenumbers versus time, which includes a summary of the kinetic components assigned to Tyr, Arg, and the different structural motifs.
© Copyright Policy
Related In: Results  -  Collection

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

fig9: The kinetics of exchange for ER is summarized in three plots. (A) Initial times of exchange. (B) Percent of unexchanged protein versus time. ER was observed to exchange 98% after 150 min. A straight line shown at the bottom of each multiexponential decay plot (A and B) is the residual of the fit with the resulting standard deviation 0.650. (C) Evolution of the absorbance at selected wavenumbers versus time, which includes a summary of the kinetic components assigned to Tyr, Arg, and the different structural motifs.
Mentions: The overall extent of exchange for each ligand is shown in Figs. 5, A and B, to 9, A and B, and is summarized in Table 1. In general, the extent of exchange agreed with the overall hydrophobic residue content for each ligand (as analyzed by the Kyte-Doolittle hydropathy sequence analysis (60)), oligomeric state, and glycosylation. The exception was AR, suggesting that the polar residues may be buried, whereas the hydrophobic residues mainly located within C-terminal end are exposed. The fastest initial exchange was observed for TGF-α: within the first 5 min of the exchange process, 50% of the ligand had exchanged. The next fastest initial exchange was HB-EGF: within the first 5 min, 40% had exchanged, probably due to the glycosylation. Although ER had the slowest initial exchange, it was also this ligand that underwent complete exchange, suggesting that the dimer species is present in small quantities and that this protein is almost completely exposed to its aqueous environment. The extent of H/D exchange for these ligands, with EGF having the lowest extent of exchange, suggests the dimer form contributed to the loss of exposure to its aqueous environment with only 40%; in contrast, epiregulin had almost complete exchange (98%). HB-EGF had a greater extent of exchange, i.e., 65%, suggesting the glycosylation affected hydration of the ligand, whereas TGF-α and AR had extents of exchange of 65% and 55%, respectively.

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