Crystallization and preliminary crystallographic analysis of the circadian clock protein KaiB from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1.
KaiB is a component of the circadian clock oscillator in cyanobacteria, which are the simplest organisms that exhibit circadian rhythms. KaiB consists of 108 amino-acid residues and has a molecular weight of 12 025 Da. KaiB and Cys-substituted KaiB mutants from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 were expressed as GST-fusion proteins in Escherichia coli, purified and crystallized. The crystals of wild-type KaiB belong to the monoclinic space group P2(1), with unit-cell parameters a = 89.6, b = 71.2, c = 106.8 A, beta = 100.1 degrees. While the native crystals diffract to 3.7 A, osmium derivatives, which show an approximately 4 A shrinkage in the b axis, diffract to 2.6 A. The crystals of the singly Cys-substituted mutant T64C with Hg, which show different morphology, diffract to 2.5 A and belong to the monoclinic space group P2, with unit-cell parameters a = 63.7, b = 33.4, c = 93.7 A, beta = 100.1 degrees. Anomalous difference Patterson maps of the Os- and Hg-derivative crystals had significant peaks in their Harker sections, suggesting that both derivatives are suitable for structure determination.
Iwase
Ryo
R
Center for Gene Research, Nagoya University, Furo, Chikusa, Nagoya 464-8602, Japan.
Imada
Katsumi
K
Hayashi
Fumio
F
Uzumaki
Tatsuya
T
Namba
Keiichi
K
Ishiura
Masahiro
M
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002940:Circadian Rhythm; D056925:Circadian Rhythm Signaling Peptides and Proteins; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D000458:Cyanobacteria / Q000737:chemistry*
D001426:Bacterial Proteins; D056925:Circadian Rhythm Signaling Peptides and Proteins; C114270:KaiB protein, cyanobacteria