Site-Saturation Mutagenesis of Leucine 134 of Bacillus licheniformis Nucleotide Exchange Factor GrpE Reveals the Importance of this Residue to the Co-chaperone Activity

doi:10.1007/s10930-010-9261-5

CNU IR > Pharmacy and Science > Dept. of Food Science & Technology > Periodical Articles > Item 310902800/25230

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Title:	Site-Saturation Mutagenesis of Leucine 134 of Bacillus licheniformis Nucleotide Exchange Factor GrpE Reveals the Importance of this Residue to the Co-chaperone Activity
Authors:	Min-Guan Lin Bo-En Chen Wan-Chi Liang Wei-Mou Chou Jiau-Hua Chen Lih-Ying Kuo Long-Liu Lin
Contributors:	食品科技系
Keywords:	Bacillus licheniformis GrpE Site-saturation mutagenesis Co-chaperone activity Temperature-induced denaturation
Date:	2010-06
Issue Date:	2012-05-22 13:53:31 (UTC+8)
Abstract:	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 α-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.
Relation:	Protein Journal 29(5)：p.365-372
Appears in Collections:	[Dept. of Food Science & Technology] Periodical Articles

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