Direct interaction between KaiA and KaiB revealed by a site-directed spin labeling electron spin resonance analysis.
In cyanobacteria, three clock proteins, KaiA, KaiB and KaiC, play essential roles in generating circadian oscillations. The interactions of these proteins change during the circadian cycle. Here, we demonstrated direct interaction between KaiA and KaiB using electron spin resonance spectroscopy. We prepared cystein (Cys)-substituted mutants of Thermosynechococcus elongatus KaiB, labeled specifically their Cys residues with spin labels and measured the ESR spectra of the labeled KaiB. We found that KaiB labeled at the 64th residue showed spectral changes in the presence of KaiA, but not in the presence of KaiC or bovine serum albumin as a negative control. KaiB labeled at the 101st residue showed no such spectral changes even in the presence of KaiA. The results suggest that KaiB interacts with KaiA in the vicinity of the 64th residue of KaiB. Further analysis demonstrated that the C-terminal clock-oscillator domain of KaiA is responsible for this interaction.
Mutoh
Risa
R
Center for Gene Research, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8602, JapanDivision of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8602, JapanDivision of Material Science (Physics), Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8602, JapanDivision of Biological Science, Graduate School of Science, Osaka University, Machikaneyama, Toyonaka, Osaka 560-0043, Japan.
Mino
Hiroyuki
H
Murakami
Reiko
R
Uzumaki
Tatsuya
T
Takabayashi
Atsushi
A
Ishii
Kentaro
K
Ishiura
Masahiro
M
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D056925:Circadian Rhythm Signaling Peptides and Proteins / Q000737:chemistry* / Q000235:genetics; D003545:Cysteine / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D009154:Mutation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D013113:Spin Labels; D013696:Temperature
D001426:Bacterial Proteins; D056925:Circadian Rhythm Signaling Peptides and Proteins; C114269:KaiA protein, cyanobacteria; C114270:KaiB protein, cyanobacteria; C114271:KaiC protein, cyanobacteria; D011994:Recombinant Proteins; D013113:Spin Labels; D003545:Cysteine