Role of solvation barriers in protein kinetic stability
Role of solvation barriers in protein kinetic stability
复制标题
DOI：
10.1016/j.jmb.2006.05.009
复制
发表时间：
2006-07-14
影响因子：
5.6
通讯作者：
Sanchez-Ruiz, Jose M.
中科院分区：
文献类型：
--
作者：
Rodriguez-Larrea, David;Minning, Stefan;Sanchez-Ruiz, Jose M.
一些感兴趣的蛋白质系统的稳定性已被证明具有动力学基础。除了明显的生物技术意义外，一些新兴的抑制淀粉样蛋白生成的分子方法侧重于增加蛋白质天然状态的动力学稳定性，这一事实强调了理解蛋白质动力学稳定性的普遍兴趣。脂肪酶是最重要的工业酶之一。在这里，我们研究了疏棉状嗜热丝孢菌脂肪酶的野生型、四种单突变体和两种高度稳定的多突变体的热变性。在所有情况下，热变性都是不可逆的、动力学控制的并且符合二态不可逆模型。这一结果表明，涉及制备高度稳定变体的新颖的以分子动力学为中心的定向进化方法是成功的，可能是因为它解决了动力学稳定性问题，特别是因为加热的分子动力学模拟可能识别出不可逆变性的过渡态中天然相互作用被破坏的区域。此外，我们发现对活化焓和熵的突变影响非常大，但动力学尿素 m 值并未伴随类似的大变化。由此我们得出结论，这些突变效应与不可逆变性过程的过渡态的某些结构特征有关，而与溶剂可及性的巨大变化无关。最近的计算研究表明至少在某些蛋白质折叠/解折叠过程中存在溶剂化/去溶剂化障碍。因此，我们提出，溶剂化屏障（由内部接触破坏和水渗透之间的异步引起）可能有助于来自T lanuginosa 的脂肪酶的动力学稳定性（并且可能也有助于其他蛋白质的动力学稳定性）。 (c) 2006 Elsevier Ltd. 保留所有权利。
The stability of several protein systems of interest has been shown to have a kinetic basis. Besides the obvious biotechnological implications, the general interest of understanding protein kinetic stability is emphasized by the fact that some emerging molecular approaches to the inhibition of amyloidogenesis focus on the increase of the kinetic stability of protein native states. Lipases are among the most important industrial enzymes. Here, we have studied the thermal denaturation of the wild-type form, four single-mutant variants and two highly stable, multiple-mutant variants of lipase from Thermomyces lanuginosa. In all cases, thermal denaturation was irreversible, kinetically controlled and conformed to the two-state irreversible mod l e. This result supports that the novel molecular-dynamics-focused, directed-evolution approach involved in the preparation of the highly stable variants is successful likely because it addresses kinetic stability and, in particular, because heated molecular dynamics simulations possibly identify regions of disrupted native interactions in the transition state for irreversible denaturation. Furthermore, we find very large mutation effects on activation enthalpy and entropy, which were not accompanied by similarly large changes in kinetic urea m-value. From this we are led to conclude that these mutation effects are associated to some structural feature of the transition state for the irreversible denaturation process that is not linked to large changes in solvent accessibility. Recent computational studies have suggested the existence of solvation/desolvation barriers in at least some protein folding/unfolding processes. We thus propose that a solvation barrier (arising from the asynchrony between breaking of internal contacts and water penetration) may contribute to the kinetic stability of lipase from T lanuginosa (and, possibly, to the kinetic stability of other proteins as well). (c) 2006 Elsevier Ltd. All rights reserved.