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Design and characterization of a multimeric DNA binding protein using Sac7d and GCN4 as templates.
Proteins. 2005 Sep 01; 60(4):617-28.P

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.

Authors+Show Affiliations

Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

16028219

Citation

Wu, Sz-Wei, et al. "Design and Characterization of a Multimeric DNA Binding Protein Using Sac7d and GCN4 as Templates." Proteins, vol. 60, no. 4, 2005, pp. 617-28.
Wu SW, Ko TP, Chou CC, et al. Design and characterization of a multimeric DNA binding protein using Sac7d and GCN4 as templates. Proteins. 2005;60(4):617-28.
Wu, S. W., Ko, T. P., Chou, C. C., & Wang, A. H. (2005). Design and characterization of a multimeric DNA binding protein using Sac7d and GCN4 as templates. Proteins, 60(4), 617-28.
Wu SW, et al. Design and Characterization of a Multimeric DNA Binding Protein Using Sac7d and GCN4 as Templates. Proteins. 2005 Sep 1;60(4):617-28. PubMed PMID: 16028219.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Design and characterization of a multimeric DNA binding protein using Sac7d and GCN4 as templates. AU - Wu,Sz-Wei, AU - Ko,Tzu-Ping, AU - Chou,Chia-Cheng, AU - Wang,Andrew H-J, PY - 2005/7/20/pubmed PY - 2006/3/15/medline PY - 2005/7/20/entrez SP - 617 EP - 28 JF - Proteins JO - Proteins VL - 60 IS - 4 N2 - 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. SN - 1097-0134 UR - https://www.unboundmedicine.com/medline/citation/16028219/Design_and_characterization_of_a_multimeric_DNA_binding_protein_using_Sac7d_and_GCN4_as_templates_ L2 - https://doi.org/10.1002/prot.20524 DB - PRIME DP - Unbound Medicine ER -