Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, IL 61801, USA.
Phys Chem Chem Phys. 2010 Apr 14;12(14):3542-9. doi: 10.1039/b925033f. Epub 2010 Feb 11.
We propose protein PTB1 : 4W as a good candidate for engineering into a downhill folder. PTB1 : 4W has a probe-dependent thermal unfolding curve and sub-millisecond T-jump relaxation kinetics on more than one time scale. Its refolding rate in denaturant is a non-linear function of denaturant concentration (curved chevron plot). Yet at high denaturant concentration its unfolding is probe-independent, and the folding kinetics can be fitted to a single exponential decay. The domain appears to fold via a mechanism between downhill folding and activated folding over several small barriers, and when denaturant is added, one of these barriers greatly increases and simplifies the observed folding to apparent two-state kinetics. We predict the simplest free energy function consistent with the thermal denaturation and kinetics experiments by using the singular value Smoluchowski dynamics (SVSD) model. PTB1 : 4W is a natural 'missing link' between downhill and activated folding. We suggest mutations that could move the protein into the downhill folding limit.
我们提出蛋白质 PTB1：4W 是一种很好的候选物，可以通过工程改造成为一种下坡折叠蛋白。PTB1：4W 具有依赖探针的热解折叠曲线和亚毫秒级 T-jump 松弛动力学，跨越多个时间尺度。其在变性剂中的复性速率是变性剂浓度的非线性函数（弯曲的楔形图）。然而，在高变性剂浓度下，其解折叠是不依赖探针的，折叠动力学可以拟合为单个指数衰减。该结构域的折叠机制似乎介于下坡折叠和激活折叠之间，跨越几个小障碍，当加入变性剂时，其中一个障碍大大增加并简化了观察到的折叠，呈现出明显的两态动力学。我们通过使用奇异值 Smoluchowski 动力学（SVSD）模型，预测了与热变性和动力学实验最一致的最简单自由能函数。PTB1：4W 是下坡折叠和激活折叠之间的天然“缺失环节”。我们提出了一些突变，可以将蛋白质推向下坡折叠极限。
