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

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-α (TGF-α), 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 × 10−3 min−1 (β-sheets and 310-helix); and for TGF-α 0.91 min−1 (Tyr), 0.27 min−1 (Arg, β-turns), and 1.41 × 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

Crystallization and preliminary X-ray characterization of full-length Chlamydomonas reinhardtii centrin

C. reinhardtii centrin, an EF-hand calcium-binding protein localized to the microtubule-organizing center of eukaryotic organisms, has been crystallized in the presence of the model peptide melittin. X-ray diffraction data were collected to 2.2 Å resolution

The structure, molecular dynamics, and energetics of centrin-melittin complex

Centrin is a calcium binding protein (CaBP) belonging to the EF-hand superfamily. As with other proteins within this family, centrin is a calcium sensor with multiple biological target proteins. We chose to study Chlamydomonas reinhardtii centrin (Crcen) and its interaction with melittin (MLT) as a model for CaBP complexes due to its amphipathic properties. Our goal was to determine the molecular interactions that lead to centrin-MLT complex formation, their relative stability, and the conformational changes associated with the interaction, when compared to the single components. For this, we determined the thermodynamic parameters that define Crcen-MLT complex formation. Two-dimensional infrared (2D IR) correlation spectroscopy were used to study the amide I', I'*, and side chain bands for 13C-Crcen, MLT, and the 13C-Crcen-MLT complex. This approach resulted in the determination of MLT's increased helicity, while centrin was stabilized within the complex. Herein we provide the first complete molecular description of centrin-MLT complex formation and the dissociation process. Also, discussed is the first structure of a CaBP-MLT complex by X-ray crystallography, which shows that MLT has a different binding orientation than previously characterized centrin-bound peptides. Finally, all of the experimental results presented herein are consistent with centrin maintaining an extended conformation while interacting with MLT. The molecular implications of these results are: (1) the recognition of hydrophobic contacts as requirements for initial binding, (2) minimum electrostatic interactions within the C-terminal end of the peptide, and (3) van der Waals interactions within MLTs N-terminal end are required for complex formation.Fil: Sosa, Liliana del Valle. Recinto Universitario de Mayagüez; Puerto Rico. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; ArgentinaFil: Alfaro, Elisa Raquel. Recinto Universitario de Mayagüez; Puerto RicoFil: Santiago, Jorge Fernando. Recinto Universitario de Mayagüez; Puerto RicoFil: Narváez, Daniel. Recinto Universitario de Mayagüez; Puerto RicoFil: Rosado, Marie Cely. Recinto Universitario de Mayagüez; Puerto RicoFil: Rodríguez, Aslin. Recinto Universitario de Mayagüez; Puerto RicoFil: Gómez, Ana María. Recinto Universitario de Mayagüez; Puerto RicoFil: Schreiter, Eric R.. Recinto Universitario de Mayagüez; Puerto RicoFil: Pastrana Ríos, Belinda. Recinto Universitario de Mayagüez; Puerto Ric