title: Gene expression and molecular characterization of a chaperone protein HtpG from Bacillus licheniformis.
abstract: Heat shock protein 90 (Hsp90/HtpG) is a highly abundant and ubiquitous ATP-dependent molecular chaperone consisting of three flexibly linked regions, an N-terminal nucleotide-binding domain, middle domain, and a C-terminal domain. Here the putative htpG gene of Bacillus licheniformis was cloned and heterologously expressed in Escherichia coli M15 cells. Native-gel electrophoresis, size exclusion chromatography, and cross-linking analysis revealed that the recombinant protein probably exists as a mixture of monomer, dimer and other oligomers in solution. The optimal conditions for the ATPase activity of B. licheniformis HtpG (BlHtpG) were 45°C and pH 7.0 in the presence of 0.5mM Mg(2+) ions. The molecular architecture of this protein was stable at higher temperatures with a transition point (Tm) of 45°C at neutral pH, whereas the Tm value was reduced to 40.8°C at pH 10.5. Acrylamide quenching experiment further indicated that the dynamic quenching constant (Ksv) of BlHtpG became larger at higher pH values. BlHtpG also experienced a significant change in the protein conformation upon the addition of ATP and organic solvents. Collectively, our experiment data may provide insights into the molecular properties of BlHtpG and identify the alteration of protein structure to forfeit the ATPase activity at alkaline conditions.
authors.affiliation: Department of Food Science and Technology, Hungkuang University, 1018 Taiwan Boulevard, Shalu District, Taichung 43302, Taiwan.
authors.forename: Hui-Fen
authors.initials: HF
authors.lastname: Lo
authors.affiliation: Department of Applied Chemistry, National Chiayi University, 300 Syuefu Road, Chiayi 60004, Taiwan.
authors.forename: Bo-En
authors.initials: BE
authors.lastname: Chen
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Nangang District, Taipei 11529, Taiwan.
authors.forename: Min-Guan
authors.initials: MG
authors.lastname: Lin
authors.affiliation: Department of Applied Chemistry, National Chiayi University, 300 Syuefu Road, Chiayi 60004, Taiwan.
authors.forename: Meng-Chun
authors.initials: MC
authors.lastname: Chi
authors.affiliation: Department of Chemistry, National Cheng Kung University, Tainan 701, Taiwan. Electronic address: catalpa0905@gmail.com.
authors.forename: Tzu-Fan
authors.initials: TF
authors.lastname: Wang
authors.affiliation: Department of Applied Chemistry, National Chiayi University, 300 Syuefu Road, Chiayi 60004, Taiwan. Electronic address: llin@mail.ncyu.edu.tw.
authors.forename: Long-Liu
authors.initials: LL
authors.lastname: Lin
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001407:Bacillus / Q000145:classification / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation; D015870:Gene Expression; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010802:Phylogeny; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013056:Spectrophotometry, Ultraviolet
chemical_list: D001426:Bacterial Proteins; D018841:HSP90 Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; C066720:HtpG protein, bacteria; D000255:Adenosine Triphosphate
keywords: ATPase activity; Alkaline pH; Bacillus licheniformis; Heat shock protein 90; Protein conformation