| Summary: | 碩士 === 國立嘉義大學 === 生化科技學系研究所 === 99 === Many molecular chaperones are heat-shock proteins (Hsp) and play an important role in the protection of the host cell against various stresses. DnaK system is a known for assisting protein folding and avoiding intracellular protein aggregation, it has two co-chaperones DnaJ and GrpE, they could accelerate the DnaK activity.
In this study two researches are conducted to invesigate the effect of specific residues of DnaK and GrpE for structural and chaperone activity in Bacillus licheniformis ATCC 14580 heat shock protein 70 (BlDnaK) system.
A DNA fragment encoding 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 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.
To further elucidate the role of leucine 134 of 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 BlDnaK, whereas the NEF-defective variants had no synergistic stimulation. Comparative analysis of the far-UV CD spectra showed that the a-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.
DnaK needs specific residues maintaining ADP-Mg2+-Pi complex at the ATP-binding cleft of protein in ATP hydrolysis. Based on the sequence homology, we have modeled the three-dimensional structure of BlDnaK, and selected five different amino acids that might be responsible for the complex. As compared with wild-type BlDnaK, site-directed mutant proteins D8A, N13D, E145A, D168A, and T173A had a dramatic reduction in their chaperone activities. Complementation test revealed that the mutant proteins lost completely the ability to rescue the temperature-sensitive growth defect of Escherichia coli dnaK756-ts. Wild-type BlDnak assisted the refolding of denatured firefly luciferase, whereas a significant decrease in this ability was observed for the mutant proteins. Simultaneous addition of BlDnaJ, BlGrpE, and NR-peptide, did not synergistically stimulate the ATPase activity of D8A, E145A, D168A and T173A. Circular dichroism spectra were nearly identical for wild-type and mutant proteins, and they, except D8A, also exhibited a similar sensitivity towards temperature-induced denaturation. These results suggest that the selected residues are critical for the proper function of BlDnaK.
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