| Research Abstract |
This project is to determine the X-ray structures of α-subunit, β_2-subunite, and α_2β_2 complex of tryptophan synthase from hyperthermophile, Pyrococcus furiosus, in order to elucidate the molecular basis of the mutual activation of the subunit interaction due to the formation of the α_2β_2 complex and the stabilization mechanism of proteins from hyperthermophile. In the period, we could succeed to determine the structures of a-subunit at 2.0 A and β_2-subunite at 2.3 A.
However, the structure of the complex was not determined at higher resolutions than 3.2 A. In this report the results of α-subunit are summarized. Although the structure of the tryptophan synthase α_2β_2 complex from Salmonella typhimurium has been already determined, this is the first report of the structure of the α-subunit alone. The α-subunit from P. furiosus (Pf-α-subunit) lacked 12 and 6 residues at the N- and C-termini, respectively, and one residue each in two loop regions as compared with that from S. typhimurium (St-α-subunit), resulting in the absence of an N-terminal helix and the shortening of a C-terminal helix. The structure of the Pf-α-subunit was essentially similar to that of the St-α-subunit in the α_2β_2 complex. The differences between both structures were discussed in connection with the higher stability of the Pf-α-subunit and the complex formation of the α and β-subunits. Calorimetric results indicated that the Pf-α-subunit has extremely high thermostability and that its higher stability is caused by an entropic effect. On the basis of structural information of both proteins, we analyzed the contributions of each stabilization factor and could conclude that hydrophobic interactions in the protein interior do not contribute to the higher stability of the Pf-α-subunit. Rather the increase in ion pairs, decrease in cavity volume, and entropic effects due to shortening the polypeptide chain play important roles in extremely high stability in Pf-α-subunit.
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