Design and characterization of a multimeric DNA binding protein using Sac7d and GCN4 as templates

Sz Wei Wu, Tzu Ping Ko, Chia Cheng Chou, Andrew H.J. Wang

Research output: Contribution to journalArticlepeer-review

3 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)617-628
Number of pages12
JournalProteins: Structure, Function and Genetics
Volume60
Issue number4
DOIs
Publication statusPublished - Sep 1 2005
Externally publishedYes

Keywords

  • Coiled coil
  • Crystal structure
  • Four helix bundle
  • Genetic engineering
  • Hyperthermophile
  • Leucine zipper
  • Transcription factor
  • X-ray diffraction

ASJC Scopus subject areas

  • Structural Biology
  • Biochemistry
  • Molecular Biology

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