Guez-Ivanier V, Bedouelle H
Groupe d'Ingénierie des Protéines (CNRS URA1129) Unité de Biochimie Cellulaire Institut Pasteur, Paris, France.
J Mol Biol. 1996 Jan 12;255(1):110-20. doi: 10.1006/jmbi.1996.0010.
The C-terminal domain (residues 320 to 419) of tyrosyl-tRNA synthetase from Bacillus stearothermophilus (Bst-TyrRS) is necessary for the binding of tRNA(Tyr) but disordered in the crystal structure. Four different criteria showed that the isolated C-terminal domain of Bst-TyrRS was at least partially folded in solution. Its spectrum of circular dichroism was compatible with a high content of secondary structure elements (56% of its residues) and these structural elements disappeared in 7.5 M urea. It was unfolded by urea along a unique transition, around 6.0 M, according to the variations in the fluorescence of its tyrosine residues and in its electrophoretic mobility through transverse gradient gels. It was denatured by heat with a temperature of half-precipitation in 30 minutes that was equal to 67.9 degrees C and close to the Bst-TyrRS one, 68.7 degrees C. Its transitions of denaturation by urea or temperature were weakly cooperative. The C-terminal domains of the TyrRSs from Escherichia coli (Eco-TyrRS) and B. stearothermophilus could be genetically exchanged without a significant loss of aminoacylation activity. A hybrid between the N-terminal domain of Bst-TyrRS and the C-terminal domain of Eco-TyrRS was precipitated by heat in 30 minutes following two transitions: 83% of the molecules were precipitated with a temperature of half-transition (51.6 degrees C) close to the Eco-TyrRS one (48.6 degrees C). The remainder was precipitated with a temperature of half-transition (65.5 degrees C) close to the Bst-TyrRS one (67.2 degrees C) or that of its N-terminal domain (68.0 degrees C). These results showed that the C-terminal domain of Eco-TyrRS could undergo a transition from a soluble active conformation to an insoluble one. The denaturations of Bst-TyrRS and of its N-terminal domain by urea occurred with two successive transitions, around 4 M and 6 M, and thus according to a complex mechanism.
嗜热栖热放线菌酪氨酰 - tRNA合成酶（Bst - TyrRS）的C末端结构域（残基320至419）对于tRNA（Tyr）的结合是必需的，但在晶体结构中是无序的。四种不同的标准表明，分离出的Bst - TyrRS的C末端结构域在溶液中至少部分折叠。其圆二色光谱与高含量的二级结构元件（其残基的56%）相符，并且这些结构元件在7.5 M尿素中消失。根据其酪氨酸残基荧光的变化以及通过横向梯度凝胶的电泳迁移率，它在约6.0 M的尿素作用下沿着独特的转变展开。它在加热时变性，30分钟内半沉淀温度为67.9℃，接近Bst - TyrRS的半沉淀温度68.7℃。其由尿素或温度引起的变性转变协同性较弱。来自大肠杆菌（Eco - TyrRS）和嗜热栖热放线菌的酪氨酰 - tRNA合成酶的C末端结构域可以进行基因交换，而不会显著丧失氨酰化活性。Bst - TyrRS的N末端结构域与Eco - TyrRS的C末端结构域之间的杂种在经过两次转变后30分钟内被加热沉淀：83%的分子在半转变温度（51.6℃）下沉淀，接近Eco - TyrRS的半转变温度（48.6℃）。其余部分在半转变温度（65.5℃）下沉淀，接近Bst - TyrRS的半转变温度（67.2℃）或其N末端结构域的半转变温度（68.0℃）。这些结果表明，Eco - TyrRS的C末端结构域可以从可溶的活性构象转变为不溶的构象。Bst - TyrRS及其N末端结构域在尿素作用下的变性发生了两次连续的转变，分别在约4 M和6 M，因此是按照复杂的机制进行的。
