Extraction of protein and RNA folding landscapes from force clamp experiments

从力夹实验中提取蛋白质和 RNA 折叠景观

• 批准号: 8118391
• 负责人: Michael Hinczewski
• 金额: $ 5.87万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-10 至 2012-08-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8118391
• 关键词: Address; Alzheimer' s Disease; Biochemical Reaction; Biological Process; Catalytic RNA; Cereals; Computer software; Coupling; DNA; Data; Defect; Disease; Equilibrium; Free Energy; Future; Goals; Heart; Ions; Kinetics; Leucine Zippers; Measurement; Measures; Methods; Molecular; Molecular Structure; Morphologic artifacts; Nature; Neurodegenerative Disorders; Parkinson Disease; Polystyrenes; Procedures; Process; Proliferating; Property; Proteins; RNA; RNA Folding; Research; Research Proposals; Role; Running; Series; Simulate; Solutions; Structure; System; Techniques; Tertiary Protein Structure; Testing; Time; Transcription Coactivator; Translating; Yeasts; design; insight; interest; laser tweezer; open source; protein folding; prototype; reconstruction; research study; simulation; single molecule; theories

DESCRIPTION (provided by applicant): Single-molecule experiments on protein and RNA folding promise unprecedented insights into the formation of molecular structure, its relationship to biological function, and diseases associated with folding defects. Such studies-like the manipulation of biomolecules in an optical tweezer force clamp-aim for a detailed, quantitative description of the folding free energy landscape and dynamics. Yet the raw data ac- cessible to the experimentalist is only an indirect measure of the protein/RNA configuration over time. The crucial theoretical challenge addressed by this research proposal is how to translate the data into a com- plete picture of the underlying folding landscape. Without a comprehensive solution to this problem there is no way to disentagle which aspects of the experimental trajectories are due to the object of interest, and which are artifacts of the measuring apparatus-in short, the full potential of single-molecule techniques cannot be realized. The proposal has the following specific goals: (1) For the case of an equilibrium optical tweezer force clamp system, devise a theoretical procedure to extract the full biomolecular free energy landscape as a function of extension, together with kinetic properties like folding rates. This will re- quire filtering out the influence of other system components- the attached DNA handles and polystyrene beads-as well as correcting for tension fluctuations and hydrodynamic interactions. (2) Implement the extraction method as an easy-to-use, open-source software package that will directly process the exper- imental time series data. The design and testing of this package will be carried out with experimental collaborators, who have recently measured optical tweezer trajectories for the folding/unfolding of a yeast leucine zipper protein domain. (3) To provide an interpretative framework and testable predictions for fu- ture force clamp experiments, run long-time, coarse-grained simulations of the T. Thermophila ribozyme in an optical tweezer. The object will be to relate the extracted free energy landscape to the precise role of counterions in the formation of the RNA tertiary structure-an aspect of the RNA folding problem that is still not quantitatively understood. Put together, the results of the proposed research will constitute a necessary step for the further progress of optical tweezer studies, and serve as a prototype for tackling measurement problems endemic in all single-molecule systems. PUBLIC HEALTH RELEVANCE: The molecular mechanisms of how proteins and RNAs fold are fundamental to understanding their roles in catalyzing biochemical reactions, and neurodegenerative diseases like Alzheimer's and Parkinson's related to misfolding. The proposed research uses theory and computation to interpret protein/RNA single-molecule experimental data, in order to extract a detailed picture of the folding energy landscape and dynamics.

描述(申请人提供)：蛋白质和RNA折叠的单分子实验有望对分子结构的形成、其与生物功能的关系以及与折叠缺陷相关的疾病提供前所未有的见解。这类研究--就像在光镊力钳中操纵生物分子一样--旨在详细、定量地描述折叠自由能的格局和动力学。然而，实验者可获得的原始数据只是对蛋白质/RNA随时间变化的结构的间接测量。这项研究提案所解决的关键理论挑战是如何将数据转换为底层折叠景观的完整图景。如果没有对这个问题的全面解决方案，就无法区分实验轨迹的哪些方面是由于感兴趣的对象，哪些是测量仪器的人工制品-简而言之，单分子技术的全部潜力无法实现。该方案有以下具体目标：(1)对于平衡的光镊力钳制系统的情况，设计了一个理论程序来提取作为扩展的函数的完整的生物分子自由能图景，以及诸如折叠速率的动力学性质。这将需要过滤掉其他系统组件--连接的DNA手柄和聚苯乙烯珠子--的影响，并校正张力波动和流体动力相互作用。(2)将提取方法实现为一个简单易用的开源软件包，可以直接处理实验时间序列数据。这一包的设计和测试将与实验合作者一起进行，他们最近测量了酵母亮氨酸拉链蛋白结构域折叠/展开的光学镊子轨迹。(3)为了给未来的力钳实验提供一个解释框架和可检验的预测，在光钳中对嗜热梭菌核酶进行了长期、粗粒度的模拟。我们的目的将是将提取的自由能图景与反离子在RNA三级结构形成中的精确作用联系起来--RNA折叠问题的一个方面仍然没有定量地了解。综上所述，拟议的研究结果将构成进一步推进光镊子研究的必要步骤，并作为解决所有单分子系统中普遍存在的测量问题的原型。 与公共健康相关：蛋白质和RNA折叠的分子机制对于了解它们在催化生化反应中的作用以及阿尔茨海默氏症和帕金森氏症等神经退行性疾病与错误折叠有关。本研究采用理论和计算相结合的方法对蛋白质/核糖核酸单分子实验数据进行解释，以求提取折叠能量格局和动力学的详细图景。