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: $ 20.4万
• 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: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=762608
• 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。折叠是至关重要的，因为结构和功能紧密相连，但是关于机制以及折叠方式如何作为物理过程仍有许多待了解。关键机械信息包含在过渡路径（TPS）中，这是分子穿过不稳定状态的短暂矩，这些状态将折叠和展开状态分开，但是由于其简洁，TPS很难检测到。这项提案抓住了我们去年对TP的首次直接观察到的一个重大突破所提供的及时机会，使用光学镊子研究在张力下研究单分子，通过利用有关TPS中编码的微观动力学的独特信息，打开新的边界来折叠。该程序侧重于3个主题：1）TP形状分析。 TP形状编码尚未研究的许多信息。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，从而设计了对扩散率的稳健测量（将动力学连接到热力学的关键参数）。我们将通过TPS中的停顿直接表征过渡状态，并测试无法验证的折叠的微观理论。我们将区分不同的TP类型，通过过渡状态反映不同的路径，从而验证了折叠的多条纹视图。2）不同分子中的TP特性。我们将扩展研究的一组分子，以确定TPS如何反映分子特性。用特定特性（屏障高度 /位置，粗糙度）设计的DNA发夹将是分子，其分子具有更复杂的折叠（RNA pseudoknots，带有，或 /结构的蛋白质），以探索折叠拓扑如何影响TP性质。我们还将通过比较具有自然选择的蛋白质中的TPS与尚无蛋白质的TPS的TPS进行比较，并将现代蛋白质中的TPS与现代蛋白质中的TPS与不同进化ERAS的祖先重建中的TPS进行比较。3）扩展测量。我们将改善时间分辨率，以使对TPS的更高精确研究比现在更多。我们还将将测量结果从平衡状态扩展到非平衡状态，从而探索具有不同特性的景观的不同部分。 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我们对折叠作为物理过程的理解。