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Investigation of the structural basis for thermostability of DNA-binding protein HU from Bacillus stearothermophilus. 1998

S Kawamura, and Y Abe, and T Ueda, and K Masumoto, and T Imoto, and N Yamasaki, and M Kimura
Laboratory of Biochemistry, Faculty of Agriculture, Kyushu University, Fukuoka 812-81, Japan.

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.

UI MeSH Term Description Entries
D008958 Models, Molecular Models used experimentally or theoretically to study molecular shape, electronic properties, or interactions; includes analogous molecules, computer-generated graphics, and mechanical structures. Molecular Models,Model, Molecular,Molecular Model
D009154 Mutation Any detectable and heritable change in the genetic material that causes a change in the GENOTYPE and which is transmitted to daughter cells and to succeeding generations. Mutations
D010450 Endopeptidases A subclass of PEPTIDE HYDROLASES that catalyze the internal cleavage of PEPTIDES or PROTEINS. Endopeptidase,Peptide Peptidohydrolases
D011487 Protein Conformation The characteristic 3-dimensional shape of a protein, including the secondary, supersecondary (motifs), tertiary (domains) and quaternary structure of the peptide chain. PROTEIN STRUCTURE, QUATERNARY describes the conformation assumed by multimeric proteins (aggregates of more than one polypeptide chain). Conformation, Protein,Conformations, Protein,Protein Conformations
D011489 Protein Denaturation Disruption of the non-covalent bonds and/or disulfide bonds responsible for maintaining the three-dimensional shape and activity of the native protein. Denaturation, Protein,Denaturations, Protein,Protein Denaturations
D002152 Calorimetry, Differential Scanning Differential thermal analysis in which the sample compartment of the apparatus is a differential calorimeter, allowing an exact measure of the heat of transition independent of the specific heat, thermal conductivity, and other variables of the sample. Differential Thermal Analysis, Calorimetric,Calorimetric Differential Thermal Analysis,Differential Scanning Calorimetry,Scanning Calorimetry, Differential
D002942 Circular Dichroism A change from planar to elliptic polarization when an initially plane-polarized light wave traverses an optically active medium. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed) Circular Dichroism, Vibrational,Dichroism, Circular,Vibrational Circular Dichroism
D004268 DNA-Binding Proteins Proteins which bind to DNA. The family includes proteins which bind to both double- and single-stranded DNA and also includes specific DNA binding proteins in serum which can be used as markers for malignant diseases. DNA Helix Destabilizing Proteins,DNA-Binding Protein,Single-Stranded DNA Binding Proteins,DNA Binding Protein,DNA Single-Stranded Binding Protein,SS DNA BP,Single-Stranded DNA-Binding Protein,Binding Protein, DNA,DNA Binding Proteins,DNA Single Stranded Binding Protein,DNA-Binding Protein, Single-Stranded,Protein, DNA-Binding,Single Stranded DNA Binding Protein,Single Stranded DNA Binding Proteins
D004795 Enzyme Stability The extent to which an enzyme retains its structural conformation or its activity when subjected to storage, isolation, and purification or various other physical or chemical manipulations, including proteolytic enzymes and heat. Enzyme Stabilities,Stabilities, Enzyme,Stability, Enzyme
D001411 Geobacillus stearothermophilus A species of GRAM-POSITIVE ENDOSPORE-FORMING BACTERIA in the family BACILLACEAE, found in soil, hot springs, Arctic waters, ocean sediments, and spoiled food products. Bacillus stearothermophilus,Bacillus thermoliquefaciens

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