| RRC ID |
47981
|
| Author |
Kinoshita E, Kinoshita-Kikuta E, Karata K, Kawano T, Nishiyama A, Yamato M, Koike T.
|
| Title |
Specific glutamic acid residues in targeted proteins induce exaggerated retardations in Phos-tag SDS-PAGE migration.
|
| Journal |
Electrophoresis
|
| 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.
|
| Volume |
38(8)
|
| Pages |
1139-1146
|
| Published |
2017-4-1
|
| DOI |
10.1002/elps.201600520
|
| PMID |
28112428
|
| MeSH |
ATPases Associated with Diverse Cellular Activities
Adenosine Triphosphatases
Binding Sites
Electrophoresis, Polyacrylamide Gel*
Electrophoretic Mobility Shift Assay*
Endopeptidase Clp
Escherichia coli Proteins
Glutamic Acid / chemistry*
Histones / analysis
Humans
Molecular Chaperones
Molecular Weight
Phosphorylation
Pyridines / pharmacology*
|
| IF |
3.081
|
| Times Cited |
0
|
|
WOS Category
|
BIOCHEMICAL RESEARCH METHODS
CHEMISTRY, ANALYTICAL
|
| Resource |
| DNA material |
Genome Network Project Human cDNA W01A114G21 (HGE045765)
W01A025I03 (HGE010195). |
