Del Valle Sosa, Liliana

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    Structural changes of ¹³C‐labelled 𝘊𝘩𝘭𝘢𝘮𝘺𝘥𝘰𝘮𝘰𝘯𝘢𝘴 centrin and melittin upon complex formation in the presence of calcium
    (2006) Del Valle Sosa, Liliana; Pastrana-Ríos, Belinda; College of Arts and Sciencies - Sciences; Ramírez, Doris; Ríos Steiner, Jorge; Department of Chemistry; Ríos Velázquez, Carlos
    Chlamydomonas centrin is an acidic, low molecular weight protein that belongs to the EF-hand superfamily of calcium binding proteins. It is an essential component of the centrosomal structure in a wide range of organisms. ¹³C-labeled Chlamydomonas centrin (¹³C-Cen) and a model peptide from bee venom, known as melittin (MLT) were studied upon complex formation. The main objective of this thesis was to study the level of protein/peptide interaction, to explore structural changes and the relative stability of centrin, mellitin, and the complex. Two sets of experiments were performed H→D exchange via attenuated total reflectance (ATR) and thermal dependence via transmission Fourier transform-infrared (FT-IR) spectroscopy. The spectral analysis was carried-out by two-dimensional correlation analysis (2D-COS). In addition, differential scanning calorimetry (DSC) experiments were performed to corroborate the existence of pre-transitions and the thermal denaturation temperature for these proteins and the complex. For the FT-IR studies, the two protein components were studied simultaneously as: the amide I’ band (1650 cm⁻¹) for MLT and the amide I*’ band (1600 cm⁻¹) for ¹³C-Cen in order to establish the structural changes during the interaction. Furthermore, the amide II band (1550 cm⁻¹) comprised of side chains and the N-H deformation mode, and the amide II’ (1450 cm⁻¹) band comprised of the N-D deformation mode for both proteins are studied to determine side chain interaction and the extent of solvation (80% for ¹³C-Cen, 100% for MLT and 100% ¹³C-Cen/MLT). The interaction within the protein complex, was driven by MLT, which was suggested from the effects observed in the α-helical motifs of centrin to a greater extent than its interaction with calcium. This effect was observed for the calorimetric studies whereby centrin pre-transition was shifted by 13 °C from 81 °C to 95 °C. Finally, these results provide the framework to study other centrin target peptides to further understand the biophysical and biochemical changes driving their interaction.