Determination of DNA-binding parameters for the Bacillus subtilis histone-like HBsu protein through introduction of fluorophores by site-directed mutagenesis of a synthetic gene.
A synthetic gene encoding the histone-like DNA-binding protein HBsu from Bacillus subtilis has been expressed in Escherichia coli. Yields of the purified protein are at least 20 mg/l culture medium. The recombinant HBsu protein is chromatographically, immunologically and functionally identical with the authentic wild-type protein. N-terminal sequencing of the purified protein confirms the fidelity of expression of the synthetic gene in E. coli. Site-directed mutagenesis of the synthetic gene was employed to replace several amino acid residues of HBsu protein with tryptophan to facilitate the determination of DNA-binding parameters by fluorescence spectroscopy. According to gel-retardation experiments, the mutant protein [Phe47----Trp]HBsu shows identical DNA binding to wild-type HBsu protein. Analysis of fluorescence binding data reveals that [Phe47----Trp]HBsu binds double-stranded DNA with a dissociation constant in the micromolar range. Computer-assisted fit of binding models to the experimental data renders positive cooperativity of binding unlikely. A dimer of [Phe47----Trp]HBsu appears to contact three or four base pairs of DNA. These results are in partial disagreement with earlier measurements on closely homologous proteins which tended to show cooperative binding and a longer DNA contact region.
Groch
N
N
Institut für Kristallographie, Freie Universität Berlin, Federal Republic of Germany.
Schindelin
H
H
Scholtz
A S
AS
Hahn
U
U
Heinemann
U
U
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics*; D001426:Bacterial Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D005798:Genes, Bacterial; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C075779:Hbsu protein, bacteria; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins