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: 2014
• 资助国家: 加拿大
• 起止时间: 2014-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=549599
• 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.

将蛋白质，DNA和RNA等生物聚合物折叠为特定结构是其多样化功能的核心，但是我们仍然无法可靠地从顺序中预测结构 - “折叠问题”仍然是现代科学中的巨大挑战。能源景观理论提供了理解折叠的基本物理框架。原则上，可以从景观的形状中预测所有折叠现象，但是景观剖面很难在实验中测量，只有少数出版。结果，景观理论通常仅在定性上使用。该项目将应用我最近开发和验证的方法，用于测量单分子中的景观谱，以证明景观可用于定量描述和预测折叠现象，从而确立景观分析作为实验生物物理学的必不可少的工具。单个DNA，RNA和蛋白质分子将在激光镊子下保持在张力下，其长度在反复展开和重新分配时以高精度测量。从这些测量值中，我们将确定能量作为分子折叠的长度的函数，从而恢复景观的形状。我们将首先测试基本的观念，即在一维景观上可以通过运动很好地描述折叠。然后，我们将研究设定折叠速度限制的“摩擦”水平，研究这可能如何取决于景观中的位置以及所形成的结构的拓扑。最后，我们将测量在结构过渡本身中所花费的时间，该时间为折叠期间发生的原本看不见的显微镜过程提供了独特的窗口。这三个特定目标被紧密整合到旨在了解有关折叠的基本过程的研究计划中，又是建立景观理论作为做出可靠，定量预测的一种方式。结果将在广泛的区域中应用，从酶功能和基因调节到由阿尔茨海默氏症和疯牛病等错误折叠蛋白引起的疾病。