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Design and characterization of a multimeric DNA binding protein using Sac7d and GCN4 as templates
Author(s) -
Wu SzWei,
Ko TzuPing,
Chou ChiaCheng,
Wang Andrew H.J.
Publication year - 2005
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.20524
Subject(s) - template , dna , characterization (materials science) , computational biology , biophysics , microbiology and biotechnology , chemistry , nanotechnology , materials science , biochemistry , biology
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. Proteins 2005. © 2005 Wiley‐Liss, Inc.