Site-saturation mutagenesis of leucine 134 of Bacillus licheniformis nucleotide exchange factor GrpE reveals the importance of this residue to the co-chaperone activity.
To elucidate the role of leucine 134 of Bacillus licheniformis nucleotide exchange factor (BlGrpE), site-saturation mutagenesis was employed to generate all possible replacements for this residue. Wild-type and mutant proteins were purified by nickel-chelated chromatography and had a molecular mass of approximately 34.5 kDa. As compared with wild-type BlGrpE, the nucleotide exchange factor (NEF) activity of L134H, L134K, L134R, L134D, L134E, L134N, L134Q, L134S, L134G and L134P was reduced by more than 96%. In vitro binding assay revealed that wild-type BlGrpE and the functional variants mainly interacted with the monomer of BlDnaK, but no such interaction was observed for the remaining mutant proteins. BlGrpE and 9 mutant proteins synergistically stimulated the ATPase activity of B. licheniformis DnaK (BlDnaK), whereas the NEF-defective variants had no synergistic stimulation. Comparative analysis of the far-UV CD spectra showed that the alpha-helical content of the inactive mutant BlGrpEs was reduced significantly with respect to wild-type protein. Moreover, the inactive mutant proteins also exhibited a more sensitivity towards the temperature-induced denaturation. Taken together, these results indicate that Leu134 might play a structural role for the proper function of BlGrpE.
Lin
Min-Guan
MG
Department of Biochemical Science and Technology, National Chiayi University, 300 University Road, Chiayi, Taiwan.
Chen
Bo-En
BE
Liang
Wan-Chi
WC
Chou
Wei-Mou
WM
Chen
Jiau-Hua
JH
Kuo
Lih-Ying
LY
Lin
Long-Liu
LL
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002942:Circular Dichroism; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007930:Leucine / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016415:Sequence Alignment; D013696:Temperature; D013816:Thermodynamics
D001426:Bacterial Proteins; C068113:GrpE protein, Bacteria; D006360:Heat-Shock Proteins; D007930:Leucine