title: The biochemical effect of Ser166 phosphorylation on Euplotes octocarinatus centrin.
abstract: Centrin is a member of the EF-hand superfamily that is phosphorylated during mitosis and is associated with alterations of contractile fibers. To obtain insight into the structural basis for the functional effects of phosphorylation, we found that the serine residue at position 166 of Euplotes octocarinatus centrin (EoCen) can be phosphorylated by protein kinase A (PKA) in the absence or presence of metal ions using (31)P-NMR spectroscopy. Cations of Ca(2+) and Tb(3+) bound to EoCen resulted in an important structural transition from a closed to an open state. EoCen in both the closed and the open state can be phosphorylated by PKA. After phosphorylation, secondary and tertiary structural changes of EoCen, mainly on its C-terminal domain (C-EoCen), were noted through circular dichroism spectroscopy, native polyacrylamide gel electrophoresis, and 2-p-toluidinylnaphthalene-6-sulfonate fluorescence. After the protein was phosphorylated, the α-helix content and the extent of the exposed hydrophobic surface on EoCen were decreased. Phosphorylated EoCen has higher affinity for the peptide melittin than nonphosphorylated EoCen. In addition, binding of melittin with phosphorylated C-EoCen was enthalpy-driven.
authors.affiliation: Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Molecular Science, Shanxi University, Taiyuan 030006, China.
authors.forename: Ya-Qin
authors.initials: YQ
authors.lastname: Zhao
authors.forename: Jun
authors.initials: J
authors.lastname: Yan
authors.forename: Jian-Bin
authors.initials: JB
authors.lastname: Chao
authors.forename: Ai-Hhua
authors.initials: AH
authors.lastname: Liang
authors.forename: Bin-Sheng
authors.initials: BS
authors.lastname: Yang
mesh_terms: D000595:Amino Acid Sequence; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry* / Q000378:metabolism*; D016812:Euplotes; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008555:Melitten / Q000378:metabolism; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011487:Protein Conformation; D015800:Protozoan Proteins / Q000737:chemistry* / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D002135:Calcium-Binding Proteins; D002868:Chromosomal Proteins, Non-Histone; D008670:Metals; D015800:Protozoan Proteins; C056608:caltractin; D008555:Melitten; D012694:Serine