The physical basis of structure formation in biomolecules: measuring energy landscapes for protein and nucleic acid folding using single-molecule force spectroscopy

生物分子结构形成的物理基础：使用单分子力谱测量蛋白质和核酸折叠的能量景观

• 批准号: 342143-2013
• 负责人: Woodside, Michael
• 金额: $ 3.79万
• 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: 2015
• 资助国家: 加拿大
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=572462
• 关键词: physical; basis; structure; formation; biomolecules

The folding of biopolymers like proteins, DNA, and RNA into specific structures lies at the heart of their diverse functionality, yet we are still unable reliably to predict structure from sequence--the "folding problem" remains a grand challenge in modern science. Energy landscape theory provides the fundamental physical framework for understanding folding. In principle, all folding phenomena can be predicted from the shape of the landscape, but landscape profiles are very difficult to measure experimentally, with only a handful ever published. As a result, landscape theory is typically used only qualitatively. This project will apply the methods I recently developed and validated for measuring landscape profiles in single molecules to demonstrate that landscapes can be used to describe and predict folding phenomena quantitatively, establishing landscape analysis as an essential tool of experimental biophysics. Single DNA, RNA, and protein molecules will be held under tension by laser tweezers and their length measured with high precision as they repeatedly unfold and refold. From these measurements we will determine the energy as a function of the length as the molecule folds, thereby recovering the shape of the landscape. We will first test the basic notion that folding can be described well in terms of motion over a one-dimensional landscape. We will then investigate the level of "friction" that sets the speed limit for folding, studying how this may depend on the position in the landscape and the topology of the structure being formed. Finally, we will measure the time spent during the structural transition itself, which provides a unique window into the otherwise invisible microscopic processes taking place during folding. These three specific aims are tightly integrated into a research program designed both to understand the fundamental processes governing folding, and to establish landscape theory as a way to make reliable, quantitative predictions. The results will have applications in a wide range of areas, from enzyme function and gene regulation to diseases caused by incorrectly-folded proteins such as Alzheimer's and mad cow disease.

蛋白质、DNA 和 RNA 等生物聚合物折叠成特定结构是其多样化功能的核心，但我们仍然无法可靠地从序列预测结构——“折叠问题”仍然是现代科学的一个巨大挑战。能量景观理论为理解折叠提供了基本的物理框架。原则上，所有折叠现象都可以从景观的形状来预测，但景观剖面很难通过实验测量，只有少数已发表。因此，景观理论通常仅用于定性。该项目将应用我最近开发和验证的用于测量单分子景观轮廓的方法，以证明景观可用于定量描述和预测折叠现象，将景观分析确立为实验生物物理学的重要工具。单个 DNA、RNA 和蛋白质分子将被激光镊子保持在张力下，并在它们反复展开和重新折叠时高精度地测量它们的长度。根据这些测量结果，我们将确定分子折叠时能量与长度的函数关系，从而恢复景观的形状。我们将首先测试一个基本概念，即折叠可以用一维景观上的运动来很好地描述。然后，我们将研究设定折叠速度限制的“摩擦”水平，研究这如何取决于景观中的位置和所形成结构的拓扑结构。最后，我们将测量结构转变本身所花费的时间，这为观察折叠过程中发生的原本看不见的微观过程提供了一个独特的窗口。这三个具体目标被紧密地整合到一个研究项目中，该项目旨在了解控制折叠的基本过程，并建立景观理论作为进行可靠的定量预测的一种方法。这些结果将在广泛的领域得到应用，从酶功能和基因调控到由错误折叠的蛋白质引起的疾病，如阿尔茨海默病和疯牛病。