Investigation of the structural basis for thermostability of DNA- binding protein HU from Bacillus stearothermophilus

Site-directed mutagenesis was used to identify amino acid residues essential for the thermostabillty of the DNA-binding protein HU from the thermophile Bacillus stearothermophilus (BstHU). Two mutants, BstHU-A27S and BstHU-V42I, in which Ala²⁷ and Val⁴² in BstHU were replaced by the corresponding amino acids Ser²⁷ and Ile⁴², respectively, in the homologue from a mesophile B. subtilis (BsuHU), were less stable than the wild-type BstHU (63.9 °C), showing T(m) values of 58.4 °C and 60.1 °C, respectively, as estimated by circular dichroism (CD) analysis at pH 7.0. The denaturation of two mutants was further characterized using differential scanning calorimetry; the T(m) values obtained by calorimetric analysis were in good agreement with those estimated by CD analysis. The results suggest that Ala²⁷ and Val⁴² are partly responsible for enhancing the thermostability of BstHU. When considered together with previous results, it is revealed that Gly¹⁵, Ala²⁷, Glu³⁴, Lys³⁸, and Val⁴² are essential for the thermostability of thermophilic protein BstHU. Moreover, five thermostabilizing mutations were simultaneously introduced into BsuHU, which resulted in a quintuple mutant with a T(m) value of 71.3 °C, which is higher than that of BstHU, and also resulted in insusceptibility to proteinase digestion.