Glycine-15 in the bend between two α-helices can explain the thermostability of DNA binding protein HU from Bacillus stearothermophilus

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 T(m) value was 54.0 °C compared to the T(m) value of 63.9 °C for BstHU. The mutants, BstHU-T13A and BstHU-T33L, were, by contrast, slightly more stable (T(m) values 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 inciting temperature by 11.8 °C (T(m) value: 60.4 °C for BsuHU-E15G) compared to the value for BsuHU (T(m): 48.6 °C). Thus, Gly¹⁵ in the HTH motif of BstHU has an important role in the thermostability of BstHU. Characterization of the structure 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.