Residues Leu52 and Leu134 are important for the structural integrity of a nucleotide exchange factor GrpE from Bacillus licheniformis.
A DNA fragment encoding Bacillus licheniformis GrpE (BlGrpE) with double mutations at codons 52 and 134 was obtained during PCR cloning. Leu52 and Leu134 in BlGrpE were individually replaced with Pro and His to generate BlGrpE-L52P and BlGrpE-L134H. BlGrpE and BlGrpE-L52P synergistically stimulated the ATPase activity of B. licheniformis DnaK (BlDnaK); however, BlGrpE-L134H and the double-mutated protein (BlGrpE-L52P/L134H) had no co-chaperone function. BlGrpE, BlGrpE-L52P, and BlGrpE-L134H mainly interacted with the monomer of BlDnaK but non-specific interaction was observed for BlGrpE-L52P/L134H. Measurement of intrinsic fluorescence revealed a significant alteration of the microenvironment of aromatic acid residues in the mutant proteins. As compared with BlGrpE, quenching of 208-nm and 222-nm signals were observed in the mutant BlGrpEs and the single-mutated proteins were more sensitive to thermal denaturation.
Liang
Wan-Chi
WC
Department of Applied Chemistry, National Chiayi University, 300 University Road, Chiayi 600, Taiwan.
Lin
Min-Guan
MG
Chou
Wei-Mou
WM
Chi
Meng-Chun
MC
Chang
Hui-Ping
HP
Lin
Long-Liu
LL
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001407:Bacillus; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D055592:Biophysical Phenomena; D002417:Cattle; D002942:Circular Dichroism; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007930:Leucine; D018832:Molecular Chaperones / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011489:Protein Denaturation; D020673:Protein Renaturation; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence
D001426:Bacterial Proteins; C068113:GrpE protein, Bacteria; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D000251:Adenosine Triphosphatases; C115251:DnaK protein, Brevibacillus choshinensis; D007930:Leucine