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 ess ential for the thermostability of the DNA-binding protein HU from the thermophile Bacillus stearothermophilus (BstHU), Two mutants, BstHU-A2 7S and BsfHU-V42I, in which Ala(27) and Val(42) in BstHU were replaced by the corresponding amino acids Ser(27) and Ile(42), respectively, i n the homologue from a mesophile B. subtilis (BsuHU), were less stable than the wild-type BstHU (63.9 degrees C), showing T-m values of 58.4 degrees C and 60.1 degrees 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 Ala(27) and Val(42) are partly responsible for enhancing the thermostability of Bs tHU, When considered together with previous results, it is revealed th at Gly(15), Ala(27), Glu(34), Lys(38), and Val(42) are essential for t he thermostability of thermophilic protein BstHU, Moreover, five therm ostabilizing mutations were simultaneously introduced into BsuHU, whic h resulted in a quintuple mutant with a T-m value of 71.3 degrees C, w hich is higher than that of BstHU, and also resulted in insusceptibili ty to proteinase digestion.