Contribution of a salt bridge to the thermostability of DNA binding protein HU from Bacillus stearothermophilus determined by site-directed mutagenesis.
We have employed site-directed mutagenesis to evaluate the contribution of the amino acid residues, Glu34, Arg37, and Lys38, to the thermostability of the thermophilic protein, Bacillus stearothermophilus DNA binding protein HU (BstHU), relative to the mesophilic homologue, Bacillus subtilis HU (BsuHU). Mutants BstHU-E34D and BstHU-K38N, in which Glu34 and Lys38 were individually replaced by the corresponding residues, Asp34 and Asn38, in BsuHU, exhibited decreased thermostabilities by 2.08 and 2.17 kJ/mol, respectively, whereas mutant BstHU-R37K with Lys instead of Arg at position 37 showed no change in thermostability. These results suggested that Glu34 and Lys38 contribute to the thermostability of BstHU by forming a possible salt bridge on the hydrophilic surface. The role of Glu34 as a salt bridge partner was corroborated by generating BstHU mutant protein BstHU-E34Q, in which the Glu residue was changed to Gln; this substitution clearly decreased the stability of the protein by 2.71 kJ/mol. The contribution of this favorable salt bridge to the thermostability was further investigated as the salt and pH dependencies of the stabilities of the wild-type BstHU, BstHU-K38N, and BstHU-E34D. As for salt dependency, the stability of the wild-type relative to those of the two mutants decreased with an increase in ionic strength, indicating that the electrostatic interaction between Glu34 and Lys38 indeed significantly contributes to the thermostability of BstHU. As for pH dependency, the difference in thermostability between the wild-type BstHU and mutant BstHU-K38N showed that the mutant protein was as stable as the wild-type protein at pH 2.0, however, at neutral pH, the mutant protein was less stable than the wild-type protein. In contrast, the difference between the melting temperatures of mutant BstHU-E34D and the wild-type did not change as a function of pH. This suggested that the Glu34 residue may play an important role in the thermostability not only as a partner in the salt bridge with Lys38, but also by stabilizing the alpha-helix from residue 18 to 35 in BstHU. On the basis of the present results, together with the previous results [Kawamura et al. (1996) Biochemistry 35, 1195-1200], three mutations, Glu15 to Gly, Asp34 to Glu, and Asn38 to Lys, were simultaneously introduced into mesophilic protein BsuHU to demonstrate their contributions to thermostability. This combination of multiple thermostabilizing mutations generated a more stable mutant protein HU, showing a Tm value of 64.5 degrees C compared to 48.6 degrees C for the wild-type BsuHU.
Kawamura
S
S
Laboratory of Biochemistry, Faculty of Agriculture, Kyushu University, Higashi-ku, Fukuoka. mkimura@agr.kyushu-u.ac.jp
Tanaka
I
I
Yamasaki
N
N
Kimura
M
M
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001411:Geobacillus stearothermophilus / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012492:Salts; D013696:Temperature
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D012492:Salts; C040330:histone-like protein HU, bacteria