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Identification of the DNA binding surface of H‐NS protein from Escherichia coli by heteronuclear NMR spectroscopy
Author(s) -
Shindo Heisaburo,
Ohnuki Ayumi,
Ginba Hiroyuki,
Katoh Etsuko,
Ueguchi Chiharu,
Mizuno Takeshi,
Yamazaki Toshimasa
Publication year - 1999
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(99)00862-5
Subject(s) - heteronuclear molecule , mutant , chemistry , oligonucleotide , dna , nuclear magnetic resonance spectroscopy , heteronuclear single quantum coherence spectroscopy , nuclear overhauser effect , binding domain , two dimensional nuclear magnetic resonance spectroscopy , binding site , biophysics , stereochemistry , crystallography , biochemistry , biology , gene
The DNA binding domain of H‐NS protein was studied with various N‐terminal deletion mutant proteins and identified by gel retardation assay and heteronuclear 2D‐ and 3D‐NMR spectroscopies. It was shown from gel retardation assay that DNA binding affinity of the mutant proteins relative to that of native H‐NS falls in the range from 1/6 to 1/25 for H‐NS 60–137 , H‐NS 70–137 and H‐NS 80–137 , whereas it was much weaker for H‐NS 91–137 . Thus, the DNA binding domain was defined to be the region from residue A80 to the C‐terminus. Sequential nuclear Overhauser effect (NOE) connectivities and those of medium ranges revealed that the region of residues Q60–R93 in mutant protein H‐NS 60–137 forms a long stretch of disordered, flexible chain, and also showed that the structure of the C‐terminal region (residues A95–Q137) in mutant H‐NS 60–137 was nearly identical to that of H‐NS 91–137 . 1 H and 15 N chemical shift perturbations induced by complex formation of H‐NS 60–137 with an oligonucleotide duplex 14‐mer demonstrated that two loop regions, i.e. residues A80–K96 and T110–A117, play an essential role in DNA binding.