Park Y C, Guez V, Bedouelle H
Unité de Biochimie Cellulaire, Institut Pasteur, 28 rue du Docteur Roux, Paris Cedex 15, 75724, France.
J Mol Biol. 1999 Feb 19;286(2):563-77. doi: 10.1006/jmbi.1998.2501.
A dense cluster of eight residues was identified at the crossing of two alpha-helices in tyrosyl-tRNA synthetase (TyrRS) from the thermophile Bacillus stearothermophilus. Its mechanism of evolution was characterized. Four residues of this cluster are not conserved in TyrRS from the mesophile Escherichia coli. The corresponding mutations were constructed in TyrRS(Delta1), a derivative of TyrRS from B. stearothermophilus in which the anticodon binding domain is deleted. Mutations I52L (i.e. Ile52 into Leu), M55L and L105V did not affect the activity of TyrRS(Delta1) in the pyrophosphate exchange reaction whereas T51P increased it. The kinetic stabilities of TyrRS(Delta1) and its mutant derivatives at 68.5 degreesC were determined from experiments of irreversible thermal precipitation. They were in the order L105V<I52L<T51P<Wild Type</=M55L; mutation I52L partially compensated L105V in these experiments whereas M55L was coupled neither to I52L nor to L105V. Mutations I52L and L105V affected the stability of the dimeric TyrRS(Delta1) at different steps of its unfolding by urea, monitored under equilibrium conditions by spectrofluorometry or size exclusion chromatography. I52L destabilized the association between the subunits even though residue Ile52 is more than 20 A away from the subunit interface. L105V destabilized the monomeric intermediate of unfolding. The two mutational pathways, going from the wild-type TyrRS(Delta1) to the I52L-L105V double mutant through each of the single mutants were not equivalent for the stability of the monomeric intermediate and for the total stability of the dimer. One pathway contained two neutral steps whereas the other pathway contained a destabilizing step followed by a stabilizing step. Mutation I52L allowed L105V along the first pathway and compensated it along the second pathway. Thus, the effects of I52L and L105V on stability depended on the structural context. The gain in activity due to T51P was at the expense of a slight destabilization.
在嗜热脂肪芽孢杆菌的酪氨酰 - tRNA合成酶（TyrRS）中，在两条α - 螺旋的交叉处鉴定出一个由八个残基组成的密集簇。对其进化机制进行了表征。该簇中的四个残基在嗜温菌大肠杆菌的TyrRS中不保守。在TyrRS(Delta1)中构建了相应的突变体，TyrRS(Delta1)是嗜热脂肪芽孢杆菌TyrRS的一种衍生物，其中反密码子结合结构域被删除。I52L（即异亮氨酸52突变为亮氨酸）、M55L和L105V突变不影响TyrRS(Delta1)在焦磷酸交换反应中的活性，而T51P突变则增强了该活性。通过不可逆热沉淀实验测定了TyrRS(Delta1)及其突变体衍生物在68.5℃下的动力学稳定性。它们的稳定性顺序为L105V < I52L < T51P < 野生型 <= M55L；在这些实验中，I52L突变部分补偿了L105V突变，而M55L突变既不与I52L突变耦合，也不与L105V突变耦合。I52L和L105V突变在尿素诱导的TyrRS(Delta1)二聚体不同解折叠步骤中影响其稳定性，通过平衡条件下的荧光光谱法或尺寸排阻色谱法进行监测。I52L突变破坏了亚基之间的缔合，尽管异亮氨酸52残基距离亚基界面超过20 Å。L105V突变破坏了解折叠单体中间体的稳定性。从野生型TyrRS(Delta1)通过每个单突变体到I52L - L105V双突变体的两条突变途径，对于单体中间体的稳定性和二聚体的总稳定性而言并不等效。一条途径包含两个中性步骤，而另一条途径包含一个去稳定步骤，随后是一个稳定步骤。I52L突变在第一条途径中允许L105V突变并在第二条途径中补偿它。因此，I52L和L105V对稳定性的影响取决于结构背景。T51P突变导致的活性增加是以轻微的去稳定为代价的。
