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. Study holds ProTherm entries: 25225, 25226, 25227 Extra Details: GrpE; Bacillus licheniformis; Site-directed mutagenesis; ATPase activity
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:55 p.m.
|Number of data points||8|
|Assays/Quantities/Protocols||Experimental Assay: Tm ; Experimental Assay: dHvH ; Derived Quantity: dTm|
|Libraries||Mutations for sequence MAEEKQNEELNEQEELNETEAETAEAEQTAAEADAPAEETQTEMLEKQLKELQERLEEKENKLLRVQADFENYKRRARLDLEAAEKYRSQRIISDLLPALDNFERALQIDPDNEQTKSLLQGMEMVHRQILEALKNEGVEQIPSVGEQFDPNMHQAVMQVEDEAYESNAVVEELQKGYKLKDRVIRPSMVKVNQ|