期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：23
页码：7183-7188
DOI：10.1073/pnas.1419490112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceEnergy landscape theory provides the conceptual foundation for describing how structures self-assemble in proteins and nucleic acids, but energy landscapes are hard to measure experimentally, which has limited quantitative applications of landscape theory. Current methods for measuring landscapes based on single-molecule folding trajectories are strongly influenced by the properties of the instrumental probe, which can confound the results. We present a new approach that avoids these limitations, based on the splitting probability that describes the likelihood of folding proceeding to completion at a given point along the reaction coordinate. We demonstrate the method using measurements of DNA hairpins as a model system, as well as computational simulations, discussing both the advantages of this approach and its limitations. Structural self-assembly in biopolymers, such as proteins and nucleic acids, involves a diffusive search for the minimum-energy state in a conformational free-energy landscape. The likelihood of folding proceeding to completion, as a function of the reaction coordinate used to monitor the transition, can be described by the splitting probability, pfold(x). Pfold encodes information about the underlying energy landscape, and it is often used to judge the quality of the reaction coordinate. Here, we show how pfold can be used to reconstruct energy landscapes from single-molecule folding trajectories, using force spectroscopy measurements of single DNA hairpins. Calculating pfold(x) directly from trajectories of the molecular extension measured for hairpins fluctuating in equilibrium between folded and unfolded states, we inverted the result expected from diffusion over a 1D energy landscape to obtain the implied landscape profile. The results agreed well with the landscapes reconstructed by established methods, but, remarkably, without the need to deconvolve instrumental effects on the landscape, such as tether compliance. The same approach was also applied to hairpins with multistate folding pathways. The relative insensitivity of the method to the instrumental compliance was confirmed by simulations of folding measured with different tether stiffnesses. This work confirms that the molecular extension is a good reaction coordinate for these measurements, and validates a powerful yet simple method for reconstructing landscapes from single-molecule trajectories.
关键词：single-molecule biophysics ; force spectroscopy ; nucleic acid folding ; protein folding ; optical tweezers
