Department of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520, United States.
J Am Chem Soc. 2011 Aug 17;133(32):12749-57. doi: 10.1021/ja204005r. Epub 2011 Jul 22.
Coiled coils are one of the most abundant protein structural motifs and widely mediate protein interactions and force transduction or sensation. They are thus model systems for protein engineering and folding studies, particularly the GCN4 coiled coil. Major single-molecule methods have also been applied to this protein and revealed its folding kinetics at various spatiotemporal scales. Nevertheless, the folding energy and the kinetics of a single GCN4 coiled coil domain have not been well determined at a single-molecule level. Here we used high-resolution optical tweezers to characterize the folding and unfolding reactions of a single GCN4 coiled coil domain and their dependence on the pulling direction. In one axial and two transverse pulling directions, we observed reversible, two-state transitions of the coiled coil in real time. The transitions equilibrate at pulling forces ranging from 6 to 12 pN, showing different stabilities of the coiled coil in regard to pulling direction. Furthermore, the transition rates vary with both the magnitude and the direction of the pulling force by greater than 1000 folds, indicating a highly anisotropic and topology-dependent energy landscape for protein transitions under mechanical tension. We developed a new analytical theory to extract energy and kinetics of the protein transition at zero force. The derived folding energy does not depend on the pulling direction and is consistent with the measurement in bulk, which further confirms the applicability of the single-molecule manipulation approach for energy measurement. The highly anisotropic thermodynamics of proteins under tension should play important roles in their biological functions.
螺旋线圈是最丰富的蛋白质结构基序之一，广泛介导蛋白质相互作用和力转导或感觉。因此，它们是蛋白质工程和折叠研究的模型系统，特别是 GCN4 螺旋线圈。主要的单分子方法也已应用于该蛋白，并揭示了其在各种时空尺度下的折叠动力学。然而，单个 GCN4 螺旋线圈结构域的折叠能和动力学尚未在单分子水平上得到很好的确定。在这里，我们使用高分辨率光镊来表征单个 GCN4 螺旋线圈结构域的折叠和展开反应及其对拉伸方向的依赖性。在一个轴向和两个横向拉伸方向上，我们实时观察到螺旋线圈的可逆、两态转变。在从 6 到 12 pN 的拉伸力范围内，这些转变达到平衡，显示出螺旋线圈在不同拉伸方向下的不同稳定性。此外，过渡速率随拉伸力的大小和方向变化超过 1000 倍，表明在机械张力下蛋白质转变具有高度各向异性和拓扑依赖性的能量景观。我们开发了一种新的分析理论来提取零力下蛋白质转变的能量和动力学。得出的折叠能不依赖于拉伸方向，与体相测量一致，进一步证实了单分子操纵方法在能量测量中的适用性。蛋白质在张力下的高度各向异性热力学应该在它们的生物学功能中发挥重要作用。
