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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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MS analysis: TOF-ES MS spectra of (A) EGF, (B) TGF-α and MALDI, (C) AR, (D) HB-EGF, and (E) ER. EGF and HB-EGF are in dimer states, whereas TGF-α, AR, and ER are in their monomeric state.
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fig2: MS analysis: TOF-ES MS spectra of (A) EGF, (B) TGF-α and MALDI, (C) AR, (D) HB-EGF, and (E) ER. EGF and HB-EGF are in dimer states, whereas TGF-α, AR, and ER are in their monomeric state.

Mentions: Although these ligands were of commercial origin, time of flight (TOF) matrix assisted laser desorption ionization (MALDI)-electrospray (ES) + MS for EGF and TGF-α and MALDI MS analyses were carried out for AR, HB-EGF, and ER to verify purity, oligomeric state, and molecular weight of each ligand sample (Fig. 2). For EGF (Fig. 2 A), a peak corresponding to the monomer form at 6,360.0 mass/charge ratio (m/z) along with a dimer at 12,720.0 m/z of this ligand is commonly observed for the recombinant form of this protein. In the case of TGF-α (Fig. 2 B), a prominent peak at 5,545.0 m/z along with a minor peak at 2,772.0 m/z for the double protonated species agreed well with the calculated molecular weight of TGF-α. For AR (Fig. 2 C), a major peak observed at 11,297.0 m/z agrees well with the expected molecular weight and a shoulder at 10,956.0 m/z, which would account for the loss of the first three amino acids in the sequence (Ser, Val, and Arg). A second peak was observed at 5,651.4 m/z and a shoulder at 5,481.4 m/z, which would account for the double protonated species. A third peak is observed for AR at 8,206.0 m/z with two minor shoulders at 8,333.6 m/z and 8,078.7 m/z of the single protonated species, possibly due to proteolytic cleavage from the Lys-N protease (based on results obtain from the Expasy peptide cutter subroutine (59))—in this case, accounting for the loss of the first 25 residues within the sequence and consequently the loss of the random coil portion found at the N-terminal end of this ligand, thus allowing the conformational analysis AR.


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)

MS analysis: TOF-ES MS spectra of (A) EGF, (B) TGF-α and MALDI, (C) AR, (D) HB-EGF, and (E) ER. EGF and HB-EGF are in dimer states, whereas TGF-α, AR, and ER are in their monomeric state.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: MS analysis: TOF-ES MS spectra of (A) EGF, (B) TGF-α and MALDI, (C) AR, (D) HB-EGF, and (E) ER. EGF and HB-EGF are in dimer states, whereas TGF-α, AR, and ER are in their monomeric state.
Mentions: Although these ligands were of commercial origin, time of flight (TOF) matrix assisted laser desorption ionization (MALDI)-electrospray (ES) + MS for EGF and TGF-α and MALDI MS analyses were carried out for AR, HB-EGF, and ER to verify purity, oligomeric state, and molecular weight of each ligand sample (Fig. 2). For EGF (Fig. 2 A), a peak corresponding to the monomer form at 6,360.0 mass/charge ratio (m/z) along with a dimer at 12,720.0 m/z of this ligand is commonly observed for the recombinant form of this protein. In the case of TGF-α (Fig. 2 B), a prominent peak at 5,545.0 m/z along with a minor peak at 2,772.0 m/z for the double protonated species agreed well with the calculated molecular weight of TGF-α. For AR (Fig. 2 C), a major peak observed at 11,297.0 m/z agrees well with the expected molecular weight and a shoulder at 10,956.0 m/z, which would account for the loss of the first three amino acids in the sequence (Ser, Val, and Arg). A second peak was observed at 5,651.4 m/z and a shoulder at 5,481.4 m/z, which would account for the double protonated species. A third peak is observed for AR at 8,206.0 m/z with two minor shoulders at 8,333.6 m/z and 8,078.7 m/z of the single protonated species, possibly due to proteolytic cleavage from the Lys-N protease (based on results obtain from the Expasy peptide cutter subroutine (59))—in this case, accounting for the loss of the first 25 residues within the sequence and consequently the loss of the random coil portion found at the N-terminal end of this ligand, thus allowing the conformational analysis AR.

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