X-ray structural analysis of tryptophan synthase a and P-subunits and its α_2β_2 complex from hyperthermophile

本课题旨在通过对嗜热性高温球菌（Pyrococcus furiosus）色氨酸合成酶α-亚基、β_2亚基和α_2β_2复合物的x射线结构的测定，阐明α_2β_2复合物形成的亚基相互激活的分子基础和嗜热性高温球菌蛋白质的稳定机制。在此期间，我们成功地确定了α亚基在2.0 A和β_2亚基在2.3 A的结构。然而，在高于3.2 A的分辨率下无法确定配合物的结构。本文对α-亚基的研究结果进行了综述。鼠伤寒沙门菌色氨酸合成酶α_2β_2复合体的结构已被确定，但α-亚基的结构尚属首次报道。与鼠伤寒沙门氏菌（St-α-亚基）相比，P. furiosus的α-亚基（Pf-α-亚基）在N端和c端分别缺失12个和6个残基，在两个环区各缺失1个残基，导致N端螺旋缺失，c端螺旋缩短。Pf-α-亚基的结构与α_2β_2配合物中St-α-亚基的结构基本相似。讨论了两种结构之间的差异，并与Pf-α-亚基的高稳定性和α和β-亚基的络合物形成有关。量热结果表明，Pf-α-亚基具有极高的热稳定性，其高稳定性是由熵效应引起的。基于这两种蛋白的结构信息，我们分析了每个稳定因子的贡献，可以得出结论，蛋白质内部的疏水相互作用并不是Pf-α-亚基更高稳定性的原因。而离子对的增加、空腔体积的减小以及多肽链的缩短所带来的熵效应才是Pf-α-亚基具有极高稳定性的重要原因。

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.