Overexpression and structural characterization of the phage T4 protein DsbA.
The double strand binding protein A (DsbA) of bacteriophage T4 is one of several viral gene products participating in transcriptional regulation. These proteins interact or associate with the host RNA polymerase core enzyme, enabling the enzyme to successively initiate transcription at different classes of viral promoters: early, middle and late. This leads to a temporally controlled expression of the T4 gene products. The DsbA binding site overlaps the late promoter region, and DsbA binding seems to intensify transcription of late genes in vitro, possibly acting as an enhancer protein (Molecular Biology of Phage T4, Karam, 1994). To further investigate the function and structure of DsbA, we overexpressed the protein in E. coli and purified it to homogeneity. Physiological functionality of the recombinant protein was shown by gel retardation experiments and by circular dichroism (CD) spectroscopy. DsbA shows strong bands in the near UV-CD spectra. The far UV-CD spectroscopy analysis shows alpha-helices to be the main secondary structure elements. This is in agreement with secondary structure predictions. A possible helix-turn-helix motif in the center of the protein could be identified. Results from crosslinking and sedimentation analyses show that DsbA forms a dimer in solution. The thermal unfolding curve fits a dimer-two-state-folding-model, and the unfolding temperature was concentration dependent. Therefore, dimerization should supply the main portion of the free energy of stabilization of deltaG0 = 42 kJ/mol.
Sieber
P
P
Lehrstuhl für Struktur und Chemie der Biopolymere, Universität Bayreuth, Germany.
Lindemann
A
A
Boehm
M
M
Seidel
G
G
Herzing
U
U
van der Heusen
P
P
Müller
R
R
Rüger
W
W
Jaenicke
R
R
Rösch
P
P
D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000502:physiology; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D015967:Gene Expression Regulation, Viral / Q000235:genetics*; D019077:Helix-Turn-Helix Motifs; D011401:Promoter Regions, Genetic / Q000235:genetics; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013696:Temperature; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry; D014158:Transcription, Genetic / Q000235:genetics; D014764:Viral Proteins / Q000737:chemistry* / Q000502:physiology
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D014764:Viral Proteins; C068357:dsbA protein, Enterobacteria phage T4; D012321:DNA-Directed RNA Polymerases