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

使用力谱直接测量单个生物分子折叠中的转变路径

• 批准号: RGPIN-2018-04673
• 负责人: Woodside, Michael
• 金额: $ 10.2万
• 依托单位: University of Alberta
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=693535
• 关键词: Direct; measurements; transition; paths; folding

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.

生物分子自组装（折叠）成复杂的结构。折叠至关重要，因为结构和功能紧密相连，但有关机制以及折叠如何作为物理过程发生的仍有很多知识需要了解。关键的机制信息包含在过渡路径（TP）中，即分子通过分隔折叠和展开状态的不稳定状态的短暂时刻，但由于过渡路径很短暂，因此很难检测到。该提案抓住了我们去年取得的重大突破所提供的及时机会——首次直接观察TP，使用光镊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，开辟了折叠的新领域。该计划重点关注 3 个主题：*****1) TP 形状分析。 TP 形状编码了许多尚未研究的信息。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，设计一种稳健的扩散率测量方法（连接动力学与热力学的关键参数）。我们将直接通过 TP 中的停顿来表征过渡态，并测试无法验证的微观折叠理论。我们将区分不同的TP类型，反映通过过渡态的不同路径，验证折叠的多路径观点。******2）不同分子中的TP特性。我们将扩大研究的分子范围，以确定 TP 如何反映分子特性。具有特定特性（势垒高度/位置、粗糙度）的 DNA 发夹将跟随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 特性。我们还将通过比较经过自然选择的蛋白质中的 TP 与未经历自然选择的工程蛋白质中的 TP，以及将现代蛋白质中的 TP 与不同进化时代的祖先重建中的 TP 进行比较，探讨进化如何在微观水平上塑造折叠。** ****3) 扩展测量。我们将提高时间分辨率，以便能够在比现在更广泛的分子中对TP进行更高精度的研究。我们还将测量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，我们将研究当施加力的轴改变时 TP 如何变化，探测势垒的变化，探索扩散率如何反映能量景观在拉力轴上的投影变化，并量化哪些拉力轴效果最好：反应坐标。*****这项工作将揭示以前未探索过的折叠方面，测试折叠理论中的基本概念，验证或证伪长期以来的假设，并巩固我们对折叠作为物理过程的理解。