Unique features of the folding landscape of a repeat protein revealed by pressure perturbation.
Unique features of the folding landscape of a repeat protein revealed by pressure perturbation.
复制标题
DOI：
10.1016/j.bpj.2010.02.044
复制
发表时间：
2010-06
影响因子：
3.4
通讯作者：
Jean-Baptiste Rouget;M. Schroer;C. Jeworrek;Matthias Pühse;J. Saldana;Yannick Bessin;M. Tolan;D. Barrick;R. Winter;C. Royer
中科院分区：
文献类型：
--
作者：
Jean-Baptiste Rouget;M. Schroer;C. Jeworrek;Matthias Pühse;J. Saldana;Yannick Bessin;M. Tolan;D. Barrick;R. Winter;C. Royer
蛋白质的体积性质提供了沿折叠反应坐标在不同状态之间的堆积和水合作用变化的信息，并且也与这些构象的能量学和动力学密切相关。这些体积特性可以通过压力摄动方法获得。在这项工作中，我们报告了使用荧光，小角度x射线散射和傅里叶变换红外光谱对Notch受体（Nank1-7）的锚蛋白结构域的高压展开研究。平衡和压力跳跃动力学荧光实验都符合压力下简单的两态折叠/展开转变，与类似分子量的蛋白质相比，展开的体积变化相当小。高压荧光、傅里叶变换红外光谱和小角度x射线散射测量表明，在很小的范围内增加尿素会导致更大的压力展开状态，而螺旋含量显著降低。这些观测结果强调了展开状态盆地的构象多样性。压力跳变荧光弛豫测量的温度依赖性表明，在低温下，折叠过渡态系综（TSE）在体积上接近折叠态，与屏障处明显的脱水一致。相反，TSE的热膨胀率与未折叠态的热膨胀率相等，表明约束折叠态热膨胀率的相互作用尚未在折叠势垒处建立。这种行为揭示了Nank1-7的TSE具有高度的可塑性。
The volumetric properties of proteins yield information about the changes in packing and hydration between various states along the folding reaction coordinate and are also intimately linked to the energetics and dynamics of these conformations. These volumetric characteristics can be accessed via pressure perturbation methods. In this work, we report high-pressure unfolding studies of the ankyrin domain of the Notch receptor (Nank1–7) using fluorescence, small-angle x-ray scattering, and Fourier transform infrared spectroscopy. Both equilibrium and pressure-jump kinetic fluorescence experiments were consistent with a simple two-state folding/unfolding transition under pressure, with a rather small volume change for unfolding compared to proteins of similar molecular weight. High-pressure fluorescence, Fourier transform infrared spectroscopy, and small-angle x-ray scattering measurements revealed that increasing urea over a very small range leads to a more expanded pressure unfolded state with a significant decrease in helical content. These observations underscore the conformational diversity of the unfolded-state basin. The temperature dependence of pressure-jump fluorescence relaxation measurements demonstrated that at low temperatures, the folding transition state ensemble (TSE) lies close in volume to the folded state, consistent with significant dehydration at the barrier. In contrast, the thermal expansivity of the TSE was found to be equivalent to that of the unfolded state, indicating that the interactions that constrain the folded-state thermal expansivity have not been established at the folding barrier. This behavior reveals a high degree of plasticity of the TSE of Nank1–7.