RESUMEN
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 ß-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 ß-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).
RESUMEN
Centrin is an EF-hand calcium-binding protein found in microtubule organizing centers of organisms ranging from algae and yeast to man. Phosphorylation in the centrin C-terminal domain occurs in mitosis and is associated with alterations in contractile fibers. To obtain insight into the structural basis for the functional effect of phosphorylation, Chlamydomonas reinhardtii centrin C-terminal domain phosphorylated at Ser167 (pCRC-C) has been produced and characterized. The structure of pCRC-C was compared to the unmodified protein by NMR spectroscopy. The effect of phosphorylation on target binding was examined for the complex of pCRC-C and a 19 residue centrin-binding fragment of Kar1. Remarkably, the efficient and selective phosphorylation by PKA was suppressed in the complex. Moreover, comparisons of NMR chemical shift differences induced by phosphorylation reveal a greater effect from phosphorylation in the context of the Kar1 complex than for the free protein. These results directly demonstrate that phosphorylation modulates the structure and biochemical activities of centrin.