Glycine-15 in the Bend between Two α-Helices Can Explain the Thermostability of DNA Binding Protein HU fromBacillus stearothermophilus

Publisher Website
Google Scholar
Abstract
On the basis of sequence comparison of thermophilic and mesophilic DNA binding protein HUs, Bacillus stearothermophilus DNA binding protein HU (BstHU) seems to gain thermostability with a change in amino acid residues present on the molecular surface. To evaluate the contribution of exchange of each amino acid to the thermostability of BstHU, we constructed three mutants, BstHU-T13A (Thr13 to Ala), BstHU-G15E (Gly15 to Glu), and BstHU-T33L (Thr33 to Leu), in which the amino acids in BstHU were changed to the corresponding ones in Bacillus subtilis DNA binding protein HU (BsuHU). Stability of the mutant proteins was determined from thermal-denaturation curves. Replacement of Gly15 located in the turn region between α1 and α2 helices (HTH motif), with Glu (BstHU-G15E), resulted in a decrease in thermostability, and the Tm value was 54.0 °C compared to the Tm value of 63.9 °C for BstHU. The mutants, BstHU-T13A and BstHU-T33L, were, by contrast, slightly more stable (Tmvalues of 67.0 and 65.6 °C for BstHU-T13A and BstHU-T33L, respectively) than the wild type. We then generated the BsuHU mutant protein BsuHU-E15G, where Glu15 in BsuHU was in turn replaced by Gly, and we analyzed the thermostability. This substitution clearly enhanced the melting temperature by 11.8 °C (Tm value: 60.4 °C for BsuHU-E15G) compared to the value for BsuHU (Tm: 48.6 °C). Thus, Gly15 in the HTH motif of BstHU has an important role in the thermostability of BstHU. Characterization of the stucture of the BstHU-G15E by 1H-NMR analysis showed that solvent accessibility of amide proton of Ala21 in the mutant was significantly increased compared with that of wild type, which means that the structure of the HTH motif in the N-terminal region in the mutant was changed to a more open conformation, thereby avoiding the interaction of Ala21 with either Ser17 by hydrogen bond or Ala11 by hydrophobic interaction.

This publication has 10 references indexed in Scilit: