Thermostabilization mechanism of proteins from thermophiles

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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.