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联系在一起的，但关于折叠的机制和折叠是如何作为一个物理过程发生的，还有很多需要了解。关键的机制信息包含在过渡路径(TPS)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TPS由于其简短而很难被检测到。这一提议抓住了我们去年取得的一项重大突破提供的及时机会，这项突破是我们对TPS的第一次直接观察，利用TPS中编码的关于微观动力学的独特信息，利用TPS中编码的关于微观动力学的独特信息，研究张力下的单分子。该节目重点关注3个主题：*1)TP形态分析。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，将其作为对能量格局的扩散搜索，设计出一种稳健的扩散系数测量方法(连接动力学和热力学的关键参数)。我们将通过TPS中的停顿直接表征过渡态，并测试无法验证的折叠的微观理论。我们将通过过渡态区分反映不同路径的不同TP类型，验证折叠的多路径观点。*2)不同分子中的TP性质。我们将扩大所研究的分子集合，以确定TPS如何反映分子特性。具有特定属性(屏障高度/位置、粗糙度)的DNA发夹将紧随其后的是具有更复杂折叠的分子(RNA假结、具有、或/结构的蛋白质)，以探索折叠拓扑如何影响TP属性。我们还将通过比较经过自然选择的蛋白质中的TP与未经过自然选择的工程蛋白质中的TP，以及通过比较不同进化时代现代蛋白质中的TP与祖先重建中的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，以便能够在比现在更广泛的分子中进行更高精度的TPS研究。我们还将把测量范围从平衡状态扩展到非平衡状态，从而探索具有不同属性的景观的不同部分。最后，我们将研究当作用力的轴线改变时，TPS如何变化，探测势垒的变化，探索扩散系数如何反映能量景观在拉动轴上投影的变化，并量化哪些拉动轴作为反应坐标工作得最好。*这项工作将揭示折叠以前未探索的方面，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.