Site-Saturation Mutagenesis of Leucine 134 of Bacillus licheniformis Nucleotide Exchange Factor GrpE Reveals the Importance of this Residue to the Co-chaperone Activity
| Autor: | Min-Guan Lin, Wan-Chi Liang, Long-Liu Lin, Jiau-Hua Chen, Wei-Mou Chou, Bo-En Chen, Lih-Ying Kuo |
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| Rok vydání: | 2010 |
| Předmět: |
Molecular Sequence Data
Mutant Bacillus Bioengineering Biology Biochemistry Analytical Chemistry Nucleotide exchange factor Bacterial Proteins Leucine Denaturation (biochemistry) Amino Acid Sequence Bacillus licheniformis Saturated mutagenesis Heat-Shock Proteins Molecular mass Circular Dichroism Organic Chemistry Temperature biology.organism_classification Molecular biology Co-chaperone Mutation Mutagenesis Site-Directed Thermodynamics Sequence Alignment |
| Zdroj: | The Protein Journal. 29:365-372 |
| ISSN: | 1875-8355 1572-3887 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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