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

将蛋白质、DNA和RNA等生物聚合物折叠成特定的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重大挑战。能量景观理论为理解折叠提供了基本的物理框架。原则上，所有的折叠现象都可以从景观的形状来预测，但是景观剖面很难通过实验来测量，只有少数几个已经发表。因此，景观理论通常只用于定性。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，以证明景观可以用来定量描述和预测折叠现象，建立景观分析作为实验生物物理学的一个重要工具。单个DNA、RNA和蛋白质分子将被激光镊子保持在张力下，并在重复展开和重新折叠时高精度测量它们的长度。从这些测量中，我们将确定能量作为分子折叠时长度的函数，从而恢复景观的形状。我们将首先测试基本概念，即折叠可以很好地描述在一维景观的运动。然后，我们将研究“摩擦”的水平，设置折叠的速度限制，研究这可能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，这为折叠过程中发生的不可见的微观过程提供了一个独特的窗口。这三个具体的目标是紧密结合到一个研究计划，旨在了解的基本过程中的折叠，并建立景观理论作为一种方式，使可靠的，定量的预测。这些结果将在广泛的领域中得到应用，从酶功能和基因调控到由不正确折叠的蛋白质引起的疾病，如阿尔茨海默氏症和疯牛病。

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.