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 language | English |
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Pages (from-to) | 617-628 |
Number of pages | 12 |
Journal | Proteins: Structure, Function and Genetics |
Volume | 60 |
Issue number | 4 |
DOIs | |
Publication status | Published - Sept 1 2005 |
Externally published | Yes |
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