title: Effects of Phosphorylation in Chlamydomonas Centrin Ser 167.
abstract: Centrin is a conserved calcium binding protein belonging to the EF-hand superfamily with two independent structural domains. This protein is found to be phosphorylated near the carboxyl terminal end. Our goal was to perform a novel comparative study of phosphorylated and unphosphorylated centrin by Fourier transform infrared (FT-IR) spectroscopy, two-dimensional correlation spectroscopy (2D-COS) analysis and differential scanning calorimetry (DSC). To achieve this goal, we have bacterially expressed, isolated, purified and phosphorylated centrin. We verified the extent of phosphorylation to be >97% for centrin by MALDI MS analysis and determined the absence of aggregated protein. The thermal denaturation temperature and ΔCp were determined to be T(m) = 112.1 °C (ΔCp = 7.8 Kcal/mole/ΔC) and T(m) = 111.0°C (ΔCp = 5.0 Kcal/mole/°C) for holo-centrin and phosphorylated centrin, respectively. We have also described the molecular dynamics leading up to the thermal denaturation of the protein: for holo-centrin the vibrational modes associated with the calcium binding sites aspartates and glutamates, loops then the arginines, followed by the structured backbone vibrational modes the α-helix at 1635 cm(-1) then β-sheet and finally the more exposed α-helix at 1650 cm(-1); while for phosphorylated centrin aspartate, glutamate and arginine, followed by the backbone associated vibrational modes α-helix (1650 cm(-1)), loop then the β-sheet (1633 cm(-1)) and finally the α-helix (1637 cm(-1)). Therefore, the effect on domain stability due to phosphorylation at Ser(167) was observed in the loops as well as the α-helix at 1650 cm(-1).
authors.affiliation: Department of Chemistry; University of Puerto Rico; Mayagüez Campus; Mayagüez, USA.
authors.forename: Zuleika
authors.initials: Z
authors.lastname: Sanoguet
authors.forename: Muriel
authors.initials: M
authors.lastname: Campbell
authors.forename: Sindia
authors.initials: S
authors.lastname: Ramos
authors.forename: Christina
authors.initials: C
authors.lastname: Seda
authors.forename: Luis Pérez
authors.initials: LP
authors.lastname: Moreno
authors.forename: Belinda
authors.initials: B
authors.lastname: Pastrana-Rios