Site-directed mutagenesis was used to identify amino acid residues essential for the thermostability 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 degreesC), showing Tm values of 58.4 degreesC and 60.1 degreesC, 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 Tm 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 Tm value of 71.3 degreesC, which is higher than that of BstHU, and also resulted in insusceptibility to proteinase digestion. Study holds ProTherm entries: 3435, 3436, 3437, 3438, 3439, 3440, 3441, 3442, 3443, 3444, 3445, 3446, 3447, 3448, 3449, 3450, 3451, 3452, 3453, 3454, 3455, 14224, 14225, 14226, 14227, 14228, 14229, 14230, 14231, 14232, 14233 Extra Details: Thermophilic protein thermostability; DNA-binding protein HU; structural;,Bacillus stearothermophilus
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:21 p.m.
|Number of data points||58|
|Proteins||DNA-binding protein HU ; DNA-binding protein HU 1 ; DNA-binding protein HU|
|Assays/Quantities/Protocols||Experimental Assay: ddG temp:48.6 C ; Experimental Assay: ddG temp:63.9 C ; Experimental Assay: Tm buffers:phosphate: 0.05 M, ionic:: ; Experimental Assay: Tm buffers:Sodium phosphate: 5 mM, ionic:NaCl: 0.2 M ; Experimental Assay: dHvH ; Derived Quantity: dTm buffers:phosphate: 0.05 M, ionic:: ; Derived Quantity: dTm buffers:Sodium phosphate: 5 mM, ionic:NaCl: 0.2 M|
|Libraries||Mutations for sequence MNKTELINAVAETSGLSKKDATKAVDAVFDSITEALRKGDKVQLIGFGNFEVRERAARKGRNPQTGEEMEIPASKVPAFKPGKALKDAVK ; Mutations for sequence MNKTELINAVAEASELSKKDATKAVDSVFDTILDALKNGDKIQLIGFGNFEVRERSARKGRNPQTGEEIEIPASKVPAFKPGKALKDAVAGK|