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其多种功能的核心，但我们仍然无法可靠地从序列中预测结构——“折叠问题”仍然是现代科学中的一个巨大挑战。能量景观理论为理解折叠提供了基本的物理框架。原则上，所有的折叠现象都可以从景观的形状来预测，但是景观的轮廓很难用实验来测量，只有少数发表过。因此，景观理论通常只被定性地使用。本项目将应用我最近开发并验证的测量单分子景观剖面的方法，以证明景观可以用于定量描述和预测折叠现象，将景观分析建立为实验生物物理学的重要工具。单个DNA、RNA和蛋白质分子将在激光镊子的张力下保持，并在它们反复展开和折叠时高精度地测量它们的长度。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。我们将首先测试一个基本概念，即折叠可以很好地描述为一维景观上的运动。然后，我们将研究设置折叠速度限制的“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其他不可见的微观过程提供了一个独特的窗口。这三个具体目标紧密地整合到一个研究项目中，旨在了解控制折叠的基本过程，并建立景观理论作为一种可靠的定量预测方法。这一结果将应用于广泛的领域，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.