Aksel Tural, Barrick Doug
Deparment of Biochemistry, Stanford University School of Medicine, Stanford, California.
T.C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, Maryland.
Biophys J. 2014 Jul 1;107(1):220-32. doi: 10.1016/j.bpj.2014.04.058.
Although progress has been made to determine the native fold of a polypeptide from its primary structure, the diversity of pathways that connect the unfolded and folded states has not been adequately explored. Theoretical and computational studies predict that proteins fold through parallel pathways on funneled energy landscapes, although experimental detection of pathway diversity has been challenging. Here, we exploit the high translational symmetry and the direct length variation afforded by linear repeat proteins to directly detect folding through parallel pathways. By comparing folding rates of consensus ankyrin repeat proteins (CARPs), we find a clear increase in folding rates with increasing size and repeat number, although the size of the transition states (estimated from denaturant sensitivity) remains unchanged. The increase in folding rate with chain length, as opposed to a decrease expected from typical models for globular proteins, is a clear demonstration of parallel pathways. This conclusion is not dependent on extensive curve-fitting or structural perturbation of protein structure. By globally fitting a simple parallel-Ising pathway model, we have directly measured nucleation and propagation rates in protein folding, and have quantified the fluxes along each path, providing a detailed energy landscape for folding. This finding of parallel pathways differs from results from kinetic studies of repeat-proteins composed of sequence-variable repeats, where modest repeat-to-repeat energy variation coalesces folding into a single, dominant channel. Thus, for globular proteins, which have much higher variation in local structure and topology, parallel pathways are expected to be the exception rather than the rule.
尽管在从多肽的一级结构确定其天然折叠方面已取得进展，但连接未折叠态和折叠态的途径的多样性尚未得到充分探索。理论和计算研究预测，蛋白质在漏斗状能量景观上通过平行途径折叠，尽管对途径多样性的实验检测一直具有挑战性。在这里，我们利用线性重复蛋白提供的高平移对称性和直接的长度变化来直接检测通过平行途径的折叠。通过比较共有锚蛋白重复蛋白（CARPs）的折叠速率，我们发现随着大小和重复数的增加，折叠速率明显增加，尽管过渡态的大小（由变性剂敏感性估计）保持不变。与球状蛋白的典型模型预期的降低相反，折叠速率随链长增加是平行途径的明确证明。这一结论不依赖于广泛的曲线拟合或蛋白质结构的结构扰动。通过全局拟合一个简单的平行伊辛途径模型，我们直接测量了蛋白质折叠中的成核和传播速率，并量化了沿每条路径的通量，为折叠提供了详细的能量景观。这一平行途径的发现不同于由序列可变重复组成的重复蛋白的动力学研究结果，在该研究中，适度的重复间能量变化将折叠合并到一个单一的主导通道中。因此，对于局部结构和拓扑结构变化大得多的球状蛋白，平行途径预计是例外而非规则。
