Design and characterization of a multimeric DNA binding protein using Sac7d and GCN4 as templates.
The protein Sac7d belongs to a class of small chromosomal proteins from the hyperthermophilic archaeon Sulfolobus acidocaldarius. Sac7d is extremely stable to heat, acid, and chemical agents. This protein is a monomer and it binds DNA without any particular sequence preference, while inducing a sharp kink in the DNA. By appending a leucine-zipper-like helical peptide derived from the yeast transcriptional activator GCN4 to the C-terminal end of Sac7d, the modified monomers (denoted S7dLZ) are expected to interact with each other via hydrophobic force to form a parallel dimer. The recombinant S7dLZ was expressed in Escherichia coli and purified by heating and ion-exchange chromatography. The formation of dimer was detected by gel-filtration chromatography and chemical cross-link. The results of surface plasmon resonance and circular dichroism experiments showed that the DNA-binding capacity was retained. Furthermore, X-ray diffraction analysis of single crystals of S7dLZ in complex with DNA decamer CCTATATAGG showed that the leucine-zipper segments of S7dLZ were associated into an antiparallel four-helix bundle. There are two DNA fragments bound to each S7dLZ tetramer in the crystal. This model works as a successful template that endows protein a new function without losing original properties.
Wu
Sz-Wei
SW
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.
Ko
Tzu-Ping
TP
Chou
Chia-Cheng
CC
Wang
Andrew H-J
AH
D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism*; D001483:Base Sequence; D050976:Basic-Leucine Zipper Transcription Factors; D001665:Binding Sites; D018360:Crystallography, X-Ray; D019641:DNA, Archaeal / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D016350:Leucine Zippers; D046911:Macromolecular Substances / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*; D016945:Sulfolobus acidocaldarius / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*; D014961:X-Ray Diffraction
D019843:Archaeal Proteins; D050976:Basic-Leucine Zipper Transcription Factors; D019641:DNA, Archaeal; D004268:DNA-Binding Proteins; C501391:GCN4 protein, S cerevisiae; D046911:Macromolecular Substances; D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C095116:Sac7 protein, Sulfolobus