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

蛋白质、DNA和RNA等生物聚合物折叠成特定结构是它们多样化功能的核心，但我们仍然无法可靠地根据序列预测结构--“折叠问题”仍然是现代科学中的一个重大挑战。能量景观理论为理解褶皱提供了基本的物理框架。原则上，所有的折叠现象都可以从风景的形状中预测出来，但风景轮廓很难通过实验测量，只有少数发表过。因此，景观理论通常只被定性地使用。这个项目将应用我最近开发和验证的方法来测量单分子中的景观剖面，以证明景观可以用于定量描述和预测折叠现象，确立景观分析作为实验生物物理学的基本工具。单个DNA、RNA和蛋白质分子将被激光镊子拉紧，当它们重复展开和重新折叠时，它们的长度将以高精度测量。根据这些测量，我们将确定能量作为分子折叠时长度的函数，从而恢复地貌的形状。我们将首先测试基本概念，即折叠可以很好地描述为一维景观上的运动。然后，我们将调查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和正在形成的结构的拓扑结构。最后，我们将测量结构转变本身所花费的时间，这为了解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。这三个特定的目标被紧密地整合到一个研究计划中，旨在了解决定折叠的基本过程，并建立景观理论作为一种做出可靠的定量预测的方法。这些结果将在广泛的领域得到应用，从酶功能和基因调控，到阿尔茨海默氏症和疯牛病等蛋白质错误折叠引起的疾病。

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.