PMID:10428473
| Citation |
Shindo, H, Ohnuki, A, Ginba, H, Katoh, E, Ueguchi, C, Mizuno, T and Yamazaki, T (1999) Identification of the DNA binding surface of H-NS protein from Escherichia coli by heteronuclear NMR spectroscopy. FEBS Lett. 455:63-9 |
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| Abstract |
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). 1H and 15N 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. |
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| Keywords |
Amino Acid Sequence; Bacterial Proteins; Base Sequence; Binding Sites; DNA/metabolism; DNA-Binding Proteins/chemistry; DNA-Binding Proteins/metabolism; Escherichia coli/metabolism; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Sequence Data; Protein Structure, Secondary |
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