title: Residues Phe103 and Phe149 are critical for the co-chaperone activity of Bacillus licheniformis GrpE.
abstract: A tryptophan-free Bacillus licheniformis nucleotide exchange factor (BlGrpE) and its Trp mutants (F70W, F103W, F149W, F70/103W, F70/149W, F103/149W and F70/103/149W) were over-expressed and purified to near homogeneity. Simultaneous addition of B. licheniformis DnaJ, NR-peptide and individual variants synergistically stimulated the ATPase activity of a recombinant DnaK (BlDnaK) from the same bacterium by 3.1-14.7-fold, which are significantly lower than the synergistic stimulation (18.9-fold) of BlGrpE. Protein-protein interaction analysis revealed that Trp mutants relevant to amino acid positions 103 and 149 lost the ability to bind BlDnaK. Circular dichroism measurements indicate that F70W displayed a comparable level of secondary structure to that of BlGrpE, and the wild-type protein and the Trp mutants as well all experienced a reversible behavior of thermal denaturation. Guanidine hydrochloride (GdnHCl)-induced unfolding transition for BlGrpE was calculated to be 1.25 M corresponding to ΔG(N-U) of 4.29 kcal/mol, whereas the unfolding transitions of mutant proteins were in the range of 0.77-1.31 M equivalent to ΔG(N-U) of 2.41-4.14 kcal/mol. Taken together, the introduction of tryptophan residue, especially at positions 103 and 149, into the primary structure of BlGrpE has been proven to be detrimental to structural integrity and proper function of the protein.
authors.affiliation: Department of Applied Chemistry, National Chiayi University, 300 Syuefu Road, Chiayi City 60004, Taiwan.
authors.forename: Min-Guan
authors.initials: MG
authors.lastname: Lin
authors.affiliation: Department of Applied Chemistry, National Chiayi University, 300 Syuefu Road, Chiayi City 60004, Taiwan.
authors.forename: Meng-Chun
authors.initials: MC
authors.lastname: Chi
authors.affiliation: Department of Applied Chemistry, National Chiayi University, 300 Syuefu Road, Chiayi City 60004, Taiwan.
authors.forename: Bo-En
authors.initials: BE
authors.lastname: Chen
authors.affiliation: Department of Chemistry, National Cheng Kung University, Tainan City 701, Taiwan.
authors.forename: Tzu-Fan
authors.initials: TF
authors.lastname: Wang
authors.affiliation: Department of Food Science and Technology, Hungkuang University, 34 Chungchie Road, Taichung City 43302, Taiwan. Electronic address: hflo@sunrise.hk.edu.tw.
authors.forename: Huei-Fen
authors.initials: HF
authors.lastname: Lo
authors.affiliation: Department of Applied Chemistry, National Chiayi University, 300 Syuefu Road, Chiayi City 60004, Taiwan. Electronic address: llin@mail.ncyu.edu.tw.
authors.forename: Long-Liu
authors.initials: LL
authors.lastname: Lin
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000737:chemistry / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D015964:Gene Expression Regulation, Bacterial; D019791:Guanidine / Q000737:chemistry; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry* / Q000235:genetics; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; C068113:GrpE protein, Bacteria; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D050505:Mutant Proteins; D014364:Tryptophan; D019791:Guanidine
keywords: Bacillus licheniformis; GrpE; Tryptophan engineering