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

Shunsuke Kawamura, Yoshito Abe, Tadashi Ueda, Kiyonari Masumoto, Taiji Imoto, Nobuyuki Yamasaki, Makoto Kimura

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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.

Original languageEnglish
Pages (from-to)19982-19987
Number of pages6
JournalJournal of Biological Chemistry
Issue number32
Publication statusPublished - Aug 7 1998

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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