Direct measurements of transition paths in the folding of single biomolecules using force spectroscopy

生物分子自组装（折叠）成复杂的结构。折叠是至关重要的，因为结构和功能是紧密相连的，但仍有很多关于机制和折叠如何作为一个物理过程发生的知识有待了解。关键的机制信息包含在过渡路径（TP）中，即分子通过分离折叠和未折叠状态的不稳定状态的短暂时刻，但由于其短暂性，TP难以检测。这个建议抓住了一个及时的机会，我们去年取得了一个重大突破，第一次直接观察TP，使用光学镊子研究张力下的单分子，通过利用TP中编码的微观动力学的独特信息，开辟了折叠的新前沿。该计划侧重于3个主题：1）TP形状的分析。TP形状编码了许多尚未被研究的信息。我们将使用TP速度来测试折叠的物理理论，作为对能量景观的扩散搜索，设计扩散率（连接动力学和热力学的关键参数）的鲁棒测量。我们将直接通过TP中的停顿来验证过渡态，并测试不可能验证的折叠微观理论。我们将区分不同的TP类型，反映通过过渡态的不同路径，验证折叠的多途径观点。2）不同分子中的TP性质。我们将扩大研究的分子集，以确定TP如何反映分子特性。具有特定特性（屏障高度/位置，粗糙度）的DNA发夹将被具有更复杂折叠的分子（RNA假结，具有，或/结构的蛋白质）所跟踪，以探索折叠拓扑结构如何影响TP特性。我们还将通过比较经历过自然选择的蛋白质中的TP与没有经历过自然选择的工程蛋白质中的TP，以及通过比较现代蛋白质中的TP与不同进化时期的祖先重建中的TP，来探索进化如何在微观水平上塑造折叠。我们将提高我们的时间分辨率，使更高精度的研究TP在更广泛的分子比现在可能的。我们还将从平衡状态扩展到非平衡状态的测量，从而探索具有不同属性的景观的不同部分。最后，我们将研究当施加力的轴改变时，TP如何改变，探测势垒的变化，探索扩散率如何反映能量景观在拉动轴上的投影变化，并量化哪些拉动轴最适合作为反应坐标。这项工作将揭示以前未探索的折叠方面，测试折叠理论的基本概念，验证或证伪长期持有的假设，并巩固我们对折叠作为一个物理过程的理解。

Biological molecules self-assemble (fold') into complex structures. Folding is critical because structure and function are tightly linked, but much remains to be learned about mechanisms and how folding occurs as a physical process. Critical mechanistic information is contained in the transition paths (TPs), the brief moments when the molecule passes through the unstable states that separate the folded and unfolded states, but TPs are hard to detect owing to their brevity. This proposal seizes a timely opportunity offered by a major breakthrough we made last yearthe first direct observation of TPs, studying single molecules under tension using optical tweezersto open a new frontier in folding by exploiting the unique information about microscopic dynamics encoded in TPs. The program focuses on 3 themes:1) Analysis of TP shapes. TP shapes encode much information that has not yet been studied. We will use TP velocities to test physical theories of folding as a diffusive search over an energy landscape, devising a robust measurement of the diffusivity (the key parameter connecting kinetics to thermodynamics). We will characterise transition states directly via pauses in TPs and test microscopic theories of folding that have been impossible to validate. We will distinguish different TP types reflecting different paths through the transition states, validating the multi-pathway view of folding.2) TP properties in different molecules. We will expand the set of molecules studied to establish how TPs reflect molecular properties. DNA hairpins engineered with specific properties (barrier height/position, roughness) will be followed by molecules with more complex folds (RNA pseudoknots, proteins with , , or / structures) to explore how fold topology affects TP properties. We will also probe how evolution shapes folding at the microscopic level by comparing TPs in proteins that have undergone natural selection to TPs in engineered proteins that have not, and by comparing TPs in modern proteins to TPs in ancestral reconstructions at different evolutionary eras.3) Expanded measurements. We will improve our time resolution to enable higher-precision studies of TPs in a wider range of molecules than now possible. We will also extend measurements from the equilibrium regime into the non-equilibrium regime, thereby exploring different parts of the landscape with different properties. Finally, we will examine how TPs change when the axis of the applied force is changed, probing changes in the barriers, exploring how the diffusivity reflects changes in the projection of the energy landscape onto the pulling axis, and quantifying which pulling axes work best as reaction coordinates.This work will reveal previously unexplored facets of folding, test basic concepts from the theory of folding, validate or falsify long-held assumptions, and solidify our understanding of folding as a physical process.