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The Impact of ATPase Activity to the Function of Chaperonin from Sulfolobus Solfataricus P2

Author: LvXinPing
Tutor: ZhuQiZhong；DongZhiYang
School: Shandong University
Course: Biochemistry and Molecular Biology
Keywords: Sulfur Sulfolobus Molecular chaperone Protein Folding Mutation
CLC: Q93
Type: Master's thesis
Year: 2009
Downloads: 42
Quote: 1
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Abstract


Ⅱ type chaperone widely present in the cytosol archaea and eukaryotes, they can be in vivo to promote correct folding of the protein polypeptide chain to effectively suppress aggregation of denatured proteins, an important role. Sulfur Sulfolobus (Sulfolobus solfatariCUS) P2 is a typical door Crenarchaeota genus Sulfolobus thermophilic archaea, has good high temperature properties, but its molecular chaperone studies have not been reported in the international arena, the laboratory first reported the cloning and expression of genes and molecular chaperones strain confirmed its molecular chaperone is composed of different subunits of rotational symmetry $ 8 ring structure, a chaperone function, found that a significant ATPase activity chaperones. But ATP on archaeal molecular chaperone function of P2 has not been any reports. In this study, Sulfur Sulfolobus P2β molecular chaperone for the study, with other molecular chaperones type Ⅱ conservative sequence alignment and spatial structure search and ATPase activity related sites by overlap extension PCR technique successfully constructed P2 chaperone Site-directed mutagenesis, the molecular chaperone Asp104 mutated to Arg104, Ala418 mutation Thr, and this mutant gene in E. coli BL21 (DE3) to achieve a high level expression of ATPase activity obtained chaperone mutants. On this basis, the mutant protein chaperone function of the nature and conduct further studies. Sulfur solfataricus P2 chaperone expression of mutant engineered bacteria induced by IPTG, the cells collected by sonication. The supernatant was broken by 75 ℃ high temperature for 30min to remove a large number of complex proteins, and then by 80% saturation of (NH4) 2SO4 salting and Resouce Q ion chromatography purified mutant obtained monomer molecular chaperones. Chaperone purified monomer in the presence of ATP and Mg2 through 5h can be polymerized into the typical configuration of a double donut octamer chaperone. Using malachite green method, at different temperatures of the two chaperone mutants ATPase measured, the results indicate that a mutant ATPase activity was lost, another increase in ATPase activity, see our design gene mutation is associated with the ATPase activity. Green fluorescent protein (GFP) as the research object, analyzes P2 chaperone for wild-type and mutant acid-denatured protein refolding role in promoting and found that the molecular chaperone ATPase activity increased exogenous GFP mutant and wild-type refolding compared with the more significant role in promoting the loss of ATPase activity does not promote GFP mutants folding. Citrate synthase for the study, analysis of the P2 chaperone heat exogenous protein aggregation, the results revealed that the exogenous protein chaperones prevent aggregation role and ATPase related. This study showed that: Sulfur solfataricus P2 chaperone 104 and 418 aspartic acid alanine is the impact of this type of archaeal chaperonin for ATP binding and hydrolysis are the key sites, they can cause mutations ATPase activity changes. After the loss of ATPase activity, P2 also lost chaperone-mediated refolding of denatured GFP fluorescence protein refolding ability; also no longer have to improve the ability of citrate synthase thermal aggregation. The ATPase activity increased chaperone mutants can better promote refolding of denatured proteins and preventing protein thermal aggregation, illustrates archaea P2β chaperone chaperone function in the exercise of ATP hydrolysis is required to provide energy for the further study of type Ⅱ The role of molecular chaperones processes and mechanisms laid a good foundation.

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