The thermostability of DNA-binding protein HU from mesophilic, thermophilic, and extreme thermophilic bacteria

摘要

Based on primary structure comparison between four highly homologous DNA-binding proteins (HUs) displaying differential thermostability, we have employed in vitro site-directed mutagenesis to decipher their thermostability mechanism at the molecular level. The contribution of the 11 amino acids that differ between the thermophilic HUBst from Bacillus stearothermophilus (T_m = 61.6 ℃) and the mesophilic HUBsu from Bacillus subtilis (T_m = 39.7 ℃) was evaluated by replacing these amino acids in HUBst with their mesophilic counterparts. Among 11 amino acids, three residues, Gly-15, Glu-34, and Val-42, which are highly conserved in the thermophilic HUs, have been found to be responsible for the thermostability of HUBst. These amino acids in combination (HUBst-G15E/E34D/V42I) reduce the thermostability of the protein (T_m = 45.1 ℃) at the level of its mesophilic homologue HUBsu, By replacing these amino acids in HUBsu with their thermophilic counterparts, the HUBsu-E15G/D34E/I42V mutant was generated with thermostability (T_m = 57.8 ℃) at the level of thermophilic HUBst. Employing the same strategy, we generated several mutants in the extremely thermophilic HUTmar from Thermotoga maritima (T_m = 80.5 ℃), and obtained data consistent with the previous results. The triplet mutant HUTmar-G15E/E34D/V42I (T_m = 35.9 ℃) converted the extremely thermophilic protein HUTmar to mesophilic. The various forms of HU proteins were overproduced in Escherichia coli, highly purified, and the thermostability of the mutants confirmed by circular dichroism spectroscopy. The results presented here were elucidated on the basis of the X-ray structure of HUBst and HUTmar (our unpublished results), and their mechanism was proposed at the molecular level. The results clearly show that three individual local interactions located at the helix-turn-helix part of the protein are responsible for the stability of HU proteins by acting cooperatively in a common mechanism for thermostability.