1994 Fiscal Year Final Research Report Summary
Thermostabilization mechanism of proteins from thermophiles
| Project/Area Number |
04044109
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| Research Category |
Grant-in-Aid for international Scientific Research
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| Allocation Type | Single-year Grants |
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| Section | Joint Research |
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| Research Institution | Institute for Protein Resaerch, Osaka University |
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Principal Investigator |
YUTANI Katsuhide Institute for Protein Research, Osaka University, たんぱく質研究所, 助教授 (90089889)
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| Co-Investigator(Kenkyū-buntansha) |
PRIVALOV Peter l The Johns Hopking University, 生物カロリメータセンター, 教授
UEDAIRA Hatsuho National Institutes of Life Engineering, 生命工学技術研究所, 室長
SUGINO Yoshinobu Kansai University of Medical School, 教養部, 教授 (00028177)
HIRAGA Kaori Institute for Protein Research, Osaka University, 蛋白質研究所, 日本学術振興会特別研
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| Project Period (FY) |
1992 – 1994
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| Keywords | Calorimetry / Thermostability / Protein denaturation / Thermodynamics / Thermophile / 疎水性相互作用 |
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| Research Abstract |
Studies on extremely thermostable proteins that have denaturation temperature of more than 100 ^OC help us to understand the stabilization mechanism of a general globule protein, since thermodynamic parameters of the unfolding(such as enthalpy, entropy, and heat capacity changes)are assumed to have the special characteristics close to 110 ^OC(for example, a common converged value), which should be confirmed by actual measurements of the parameters at that temperature using extremely thermostable proteins. Each of genes cording sequence of alpha-amylase, pyroglutamine peptidase (PGP), and methionine aminopeptidase (MAP)from superthermophile, Pyrococcus furiosus was inserted in an expression vector for Escherichia coli. The three proteins expressed in a mesophile had properties similar to those observed in the native proteins. The denaturation temperature of each purified protein was found to be more than 100 ^OC by calorimetry. Thermodynamic parameters of unfolding for the three extremely thermostable proteins were obtained using a scanning calorimeter, DASM4 at various pHs. Their values did not coincide with those estimated from methophilic proteins, suggesting that there are some problems for the extrapolation to 110 ^OC.PGP was found to be a tetramer protein consisting of two dimmers with an inter-molecular disulfide bond(due to Cys 118). From comparison of calorimetric results of the wild-type PGP with those of the mutant protein(C118S)substituted by Ser at Cys118, we found that the wild-type protein was greatly stabilized by the intermolecular disulfide bond, although a disulfide bond which might be broken in high temperature has never seen in thermophilic proteins. On the other hands, kinetic studies of unfolding and refolding of PGP indicated that thermostabilization of the protein is caused by-extremely lowering the unfolding rate.
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