Nasedkin Alexandr, Marcellini Moreno, Religa Tomasz L, Freund Stefan M, Menzel Andreas, Fersht Alan R, Jemth Per, van der Spoel David, Davidsson Jan
Department of Chemistry-Ångström laboratory, Uppsala University, Box 523, SE-75110 Uppsala, Sweden.
Uppsala Center for Computational Chemistry, Science for Life Laboratory, Department of Cell and Molecular Biology, Uppsala University, Box 596, SE-75124 Uppsala, Sweden.
PLoS One. 2015 May 6;10(5):e0125662. doi: 10.1371/journal.pone.0125662. eCollection 2015.
The folding and unfolding of protein domains is an apparently cooperative process, but transient intermediates have been detected in some cases. Such (un)folding intermediates are challenging to investigate structurally as they are typically not long-lived and their role in the (un)folding reaction has often been questioned. One of the most well studied (un)folding pathways is that of Drosophila melanogaster Engrailed homeodomain (EnHD): this 61-residue protein forms a three helix bundle in the native state and folds via a helical intermediate. Here we used molecular dynamics simulations to derive sample conformations of EnHD in the native, intermediate, and unfolded states and selected the relevant structural clusters by comparing to small/wide angle X-ray scattering data at four different temperatures. The results are corroborated using residual dipolar couplings determined by NMR spectroscopy. Our results agree well with the previously proposed (un)folding pathway. However, they also suggest that the fully unfolded state is present at a low fraction throughout the investigated temperature interval, and that the (un)folding intermediate is highly populated at the thermal midpoint in line with the view that this intermediate can be regarded to be the denatured state under physiological conditions. Further, the combination of ensemble structural techniques with MD allows for determination of structures and populations of multiple interconverting structures in solution.
蛋白质结构域的折叠与去折叠显然是一个协同过程，但在某些情况下已检测到瞬时中间体。此类（去）折叠中间体在结构研究方面具有挑战性，因为它们通常寿命不长，而且其在（去）折叠反应中的作用常常受到质疑。研究最为深入的（去）折叠途径之一是果蝇同源异型域蛋白（EnHD）的途径：这种由61个残基组成的蛋白质在天然状态下形成三螺旋束，并通过螺旋中间体进行折叠。在此，我们利用分子动力学模拟得出EnHD在天然、中间和未折叠状态下的样本构象，并通过与四个不同温度下的小角/广角X射线散射数据进行比较，选择了相关的结构簇。利用核磁共振光谱法测定的剩余偶极耦合对结果进行了验证。我们的结果与先前提出的（去）折叠途径高度吻合。然而，结果还表明，在整个研究的温度区间内，完全未折叠状态仅占一小部分，并且（去）折叠中间体在热中点处高度富集，这与该中间体可被视为生理条件下的变性状态这一观点一致。此外，将整体结构技术与分子动力学模拟相结合，能够确定溶液中多个相互转化结构的结构和丰度。
