Characterization of the transcriptional activator CBF1 from Arabidopsis thaliana. Evidence for cold denaturation in regions outside of the DNA binding domain.
A transcriptional activator, CBF1, from Arabidopsis thaliana, which has the AP2 domain for DNA binding and regulates the cold acclimation response, was overexpressed in Escherichia coli, purified, and characterized. Analyses of the interaction between CBF1 and the C-repeat/dehydration-responsive element by fluorescence measurement showed that CBF1 binds to C-repeat/dehydration-responsive element as a monomer irrespective of the temperature. CD spectra of the intact and truncated CBF1 proteins (1-213, 41-213, 41-157, and 41-146) were measured to examine the temperature-dependent changes of the secondary structure of CBF1. The results suggested that the CBF1 protein has regions exhibiting reversible cold denaturation in the range between 30 and -5 degrees C and also has a region exhibiting thermal denaturation between 40 and 60 degrees C. This cold denaturation occurred in both the N-terminal and acidic regions. The thermal denaturation occurred in the region encompassing the AP2 domain. The difference between the retention time of CBF1 at 4 degrees C and that at 25 degrees C in gel filtration, and the decrease of the sedimentation coefficient, s20,w, caused by the temperature change from 25 to 3 degrees C, strongly suggested that the cold denaturation was accompanied by the extension of the molecule. The possible cold denaturation observed here might be a physiologically important structural response of CBF1 to cold stress.
Kanaya
E
E
Biomolecular Engineering Research Institute, 6-2-3, Furuedai, Suita, Osaka 565-0874, Japan. ekanaya@beri.co.jp
Nakajima
N
N
Morikawa
K
K
Okada
K
K
Shimura
Y
Y
D017360:Arabidopsis; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D002942:Circular Dichroism; D003080:Cold Temperature; D004247:DNA / Q000378:metabolism*; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D018257:Helix-Loop-Helix Motifs; D011487:Protein Conformation; D011489:Protein Denaturation; D029701:Saccharomyces cerevisiae Proteins; D012697:Serine Endopeptidases / Q000378:metabolism
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; C064042:CBF1 protein, S cerevisiae; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D012697:Serine Endopeptidases; C036813:lysyl endopeptidase