title: Specific glutamic acid residues in targeted proteins induce exaggerated retardations in Phos-tag SDS-PAGE migration.
abstract: We describe two unique proteins, Escherichia coli ClpX and human histone H2A, that show extremely retarded migrations relative to their molecular weights in Phos-tag SDS-PAGE, despite being nonphosphorylated. Although ClpX separated into multiple migration bands in Phos-tag gels, the separation was not due to phosphorylation. The N-terminal 47-61 region of ClpX was responsible for producing multiple phosphorylation-independent structural variants, even under denaturing conditions, and some of these variants were detected as highly up-shifted bands. By systematic Ala-scanning mutation analysis in the N-47-61 region, we concluded that the Glu-51 or Glu-54 residue was responsible for the appearance of exaggerated mobility-shifting bands. Histone H2A showed a much slower migration in Phos-tag gels in comparison with other major histones having similar molecular weights, and we found that the Glu-62 or Glu-65 residue caused the retarded migration. In addition, Phos-tag SDS-PAGE permitted us to detect a shift in the mobility of the phosphorylated form of histone H2A from that of the nonphosphorylated one. This is the first report showing that exaggerated retardation in the migration of a certain protein in Phos-tag SDS-PAGE is induced by interactions between the Phos-tag molecule and the carboxylate group of a specific Glu residue on the target.
authors.affiliation: Department of Functional Molecular Science, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
authors.forename: Eiji
authors.initials: E
authors.lastname: Kinoshita
authors.affiliation: Department of Functional Molecular Science, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
authors.forename: Emiko
authors.initials: E
authors.lastname: Kinoshita-Kikuta
authors.affiliation: Department of Genetics, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan.
authors.forename: Kiyonobu
authors.initials: K
authors.lastname: Karata
authors.affiliation: Department of Functional Molecular Science, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
authors.forename: Toshiki
authors.initials: T
authors.lastname: Kawano
authors.affiliation: Department of Functional Molecular Science, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
authors.forename: Atsuhiro
authors.initials: A
authors.lastname: Nishiyama
authors.affiliation: Department of Functional Molecular Science, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
authors.forename: Morihisa
authors.initials: M
authors.lastname: Yamato
authors.affiliation: Department of Functional Molecular Science, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
authors.forename: Tohru
authors.initials: T
authors.lastname: Koike
mesh_terms: D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D049071:Endopeptidase Clp; D029968:Escherichia coli Proteins; D018698:Glutamic Acid / Q000737:chemistry*; D006657:Histones / Q000032:analysis; D006801:Humans; D018832:Molecular Chaperones; D008970:Molecular Weight; D010766:Phosphorylation; D011725:Pyridines / Q000494:pharmacology*
chemical_list: C514153:1,3-bis(bis(pyridin-2-ylmethyl)amino)propan-2-ol; D029968:Escherichia coli Proteins; D006657:Histones; D018832:Molecular Chaperones; D011725:Pyridines; D018698:Glutamic Acid; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; C088072:ClpX protein, E coli; D000074183:ATPases Associated with Diverse Cellular Activities
keywords: Affinity electrophoresis; ClpX; Glutamic acid; Histone; Phos-tag SDS-PAGE