Factors Defining Effects of Macromolecular Crowding on Protein Stability: An in Vitro/in Silico Case Study Using Cytochrome c
Factors Defining Effects of Macromolecular Crowding on Protein Stability: An in Vitro/in Silico Case Study Using Cytochrome c
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DOI：
10.1021/bi100578x
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发表时间：
2010-08-10
期刊：
影响因子：
2.9
通讯作者：
Wittung-Stafshede, Pernilla
中科院分区：
文献类型：
--
作者：
Christiansen, Alexander;Wang, Qian;Wittung-Stafshede, Pernilla
之前对两个单域蛋白质的实验表明，大分子拥挤可以显着稳定在热扰动下，并调节自然态的结构和形状。为了评估这一点的普遍性，我们在这里测试了合成拥挤剂对细胞色素c的影响，细胞色素c是一种小的单域蛋白。利用远紫外圆二色谱(CD)，我们发现加入Ficoll或葡聚糖(0-400 mg/mL，pH 7)对细胞色素c的二级结构没有影响。热实验显示Ficoll 70和葡聚糖70具有稳定作用(5-10摄氏度)；这种作用因低水平盐酸胍(GuHCl)的存在而增强，从而破坏蛋白质的稳定。当使用较小的葡聚糖，葡聚糖40时，热效应更大(10-20摄氏度)。在计算机分析中，对细胞色素c使用基于结构的(类GO)相互作用，与体外热力学数据非常吻合，也符合标度粒子理论。对一系列Crowder大小和形状的模拟表明，Crowder越小，对细胞色素c折叠状态稳定性的有利影响就越大。结合以前的数据，我们得出结论，蛋白质的大小、稳定性、构象延展性和折叠路线，以及Crowder的大小和形状，是调节大分子拥挤对蛋白质的净影响的关键因素。
Previous experiments with two single-domain proteins showed that macromolecular crowding can stabilize dramatically toward heat perturbation and modulate native-state structure and shape. To assess the generality of this, we here tested the effects of the synthetic crowding agents on cytochrome c, a small single-domain protein. Using far-UV circular dichroism (CD), we discovered that there is no effect on cytochrome c's secondary structure upon addition of Ficoll or dextran (0-400 mg/mL, pH 7). Thermal experiments revealed stabilizing effects (5-10 degrees C) of Ficoll 70 and dextran 70; this effect was enhanced by the presence of low levels of guanidine hydrochloride (GuHCl) that destabilize the protein. When using a smaller dextran, dextran 40, the thermal effects were larger (10-20 degrees C). In silico analysis, using structure-based (Go-like) interactions for cytochrome c, is in excellent agreement with the in vitro thermodynamic data and also agrees with scaled particle theory. Simulations of a range of crowder size and shape demonstrated that the smaller the crowder the larger the favorable effect on cytochrome c's folded-state stability. Together with previous data, we conclude that protein size, stability, conformational malleability, and folding routes, as well as crowder size and shape, are key factors that modulate the net effect of macromolecular crowding on proteins.