Godoy-Ruiz Raquel, Ariza Fernando, Rodriguez-Larrea David, Perez-Jimenez Raul, Ibarra-Molero Beatriz, Sanchez-Ruiz Jose M
Departamento de Quimica Fisica, Facultad de Ciencias, 18071-Granada, Spain.
J Mol Biol. 2006 Oct 6;362(5):966-78. doi: 10.1016/j.jmb.2006.07.065. Epub 2006 Jul 31.
It appears plausible that natural selection constrains, to some extent at least, the stability in many natural proteins. If, during protein evolution, stability fluctuates within a comparatively narrow range, then mutations are expected to be fixed with frequencies that reflect mutational effects on stability. Indeed, we recently reported a robust correlation between the effect of 27 conservative mutations on the thermodynamic stability (unfolding free energy) of Escherichia coli thioredoxin and the frequencies of residues occurrences in sequence alignments. We show here that this correlation likely implies a lower limit to thermodynamic stability of only a few kJ/mol below the unfolding free energy of the wild-type (WT) protein. We suggest, therefore, that the correlation does not reflect natural selection of thermodynamic stability by itself, but of some other factor which is linked to thermodynamic stability for the mutations under study. We propose that this other factor is the kinetic stability of thioredoxin in vivo, since( i) kinetic stability relates to irreversible denaturation, (ii) the rate of irreversible denaturation in a crowded cellular environment (or in a harsh extracellular environment) is probably determined by the rate of unfolding, and (iii) the half-life for unfolding changes in an exponential manner with activation free energy and, consequently, comparatively small free energy effects can have deleterious consequences for kinetic stability. This proposal is supported by the results of a kinetic study of the WT form and the 27 single-mutant variants of E. coli thioredoxin based on the global analyses of chevron plots and equilibrium unfolding profiles determined from double-jump unfolding assays. This kinetic study suggests, furthermore, one of the factors that may contribute to the high activation free energy for unfolding in thioredoxin (required for kinetic stability), namely the energetic optimization of native-state residue environments in regions, which become disrupted in the transition state for unfolding.
看起来似乎合理的是，自然选择至少在一定程度上限制了许多天然蛋白质的稳定性。如果在蛋白质进化过程中，稳定性在相对较窄的范围内波动，那么预计突变会以反映突变对稳定性影响的频率被固定下来。事实上，我们最近报道了27个保守突变对大肠杆菌硫氧还蛋白热力学稳定性（解折叠自由能）的影响与序列比对中残基出现频率之间存在很强的相关性。我们在此表明，这种相关性可能意味着热力学稳定性的下限仅比野生型（WT）蛋白的解折叠自由能低几个kJ/mol。因此，我们认为这种相关性本身并不反映对热力学稳定性的自然选择，而是反映了与所研究突变的热力学稳定性相关的其他因素。我们提出这个其他因素是硫氧还蛋白在体内的动力学稳定性，因为（i）动力学稳定性与不可逆变性有关，（ii）在拥挤的细胞环境（或恶劣的细胞外环境）中不可逆变性的速率可能由解折叠速率决定，并且（iii）解折叠的半衰期随活化自由能呈指数变化，因此，相对较小的自由能效应可能对动力学稳定性产生有害影响。基于从双跳跃解折叠测定确定的V形曲线和平衡解折叠谱的全局分析，对大肠杆菌硫氧还蛋白的WT形式和27个单突变变体进行的动力学研究结果支持了这一观点。此外，这项动力学研究表明了可能导致硫氧还蛋白解折叠具有高活化自由能（这是动力学稳定性所必需的）的因素之一，即天然态残基环境在解折叠过渡态中会被破坏的区域中的能量优化。
