Truhlar Stephanie M E, Agard David A
Graduate Program in Chemistry and Chemical Biology, University of California, San Francisco 94143-2240, USA.
Proteins. 2005 Oct 1;61(1):105-14. doi: 10.1002/prot.20525.
Most secreted bacterial proteases, including alpha-lytic protease (alphaLP), are synthesized with covalently attached pro regions necessary for their folding. The alphaLP folding landscape revealed that its pro region, a potent folding catalyst, is required to circumvent an extremely large folding free energy of activation that appears to be a consequence of its unique unfolding transition. Remarkably, the alphaLP native state is thermodynamically unstable; a large unfolding free energy barrier is solely responsible for the persistence of its native state. Although alphaLP folding is well characterized, the structural origins of its remarkable folding mechanism remain unclear. A conserved beta-hairpin in the C-terminal domain was identified as a structural element whose formation and positioning may contribute to the large folding free energy barrier. In this article, we characterize the folding of an alphaLP variant with a more favorable beta-hairpin turn conformation (alphaLP(beta-turn)). Indeed, alphaLP(beta-turn) pro region-catalyzed folding is faster than that for alphaLP. However, instead of accelerating spontaneous folding, alphaLP(beta-turn) actually unfolds more slowly than alphaLP. Our data support a model where the beta-hairpin is formed early, but its packing with a loop in the N-terminal domain happens late in the folding reaction. This tight packing at the domain interface enhances the kinetic stability of alphaLP(beta-turn), to nearly the same degree as the change between alphaLP and a faster folding homolog. However, alphaLP(beta-turn) has impaired proteolytic activity that negates the beneficial folding properties of this variant. This study demonstrates the evolutionary limitations imposed by the simultaneous optimization of folding and functional properties.
大多数分泌型细菌蛋白酶，包括α-溶细胞蛋白酶（αLP），在合成时都带有与其折叠所需的共价连接的前体区域。αLP的折叠态势表明，其前体区域作为一种强大的折叠催化剂，对于规避因独特的去折叠转变而产生的极大的折叠活化自由能是必需的。值得注意的是，αLP的天然状态在热力学上是不稳定的；一个大的去折叠自由能屏障是其天然状态得以持续存在的唯一原因。尽管αLP的折叠已得到充分表征，但其非凡折叠机制的结构起源仍不清楚。在C端结构域中一个保守的β-发夹被确定为一种结构元件，其形成和定位可能有助于形成大的折叠自由能屏障。在本文中，我们对具有更有利的β-发夹转折构象的αLP变体（αLP（β-转折））的折叠进行了表征。实际上，αLP（β-转折）的前体区域催化的折叠比αLP更快。然而，αLP（β-转折）并没有加速自发折叠，实际上它的去折叠速度比αLP更慢。我们的数据支持这样一个模型，即β-发夹在早期形成，但其与N端结构域中的一个环的堆积在折叠反应后期才发生。在结构域界面处的这种紧密堆积增强了αLP（β-转折）的动力学稳定性，其程度与αLP和一个折叠速度更快的同源物之间的变化几乎相同。然而，αLP（β-转折）的蛋白水解活性受损，这抵消了该变体有益的折叠特性。这项研究证明了在折叠和功能特性同时优化时所受到的进化限制。
