Single Molecule Analysis of Protein Folding Energy Landscapes

蛋白质折叠能量景观的单分子分析

• 批准号: 0646565
• 负责人: Todd Krauss
• 金额: $ 36万
• 依托单位: University of Rochester
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0646565&HistoricalAwards=false
• 关键词: Single; Molecule; Analysis; Protein; Folding

This project contains an integrated research and educational plan involving the mentoring, education, and training of undergraduate and graduate students in biophysical research. The goal of the project is to understand the molecular complexity of cytochrome c (cyt c) folding, providing data vital for characterization of energy landscapes for cyt c. The measurements will test two fundamental hypotheses relevant to understanding protein folding: 1) The extent and distribution of helical propensities of the amino acid sequences play important roles in determining the stability, population, and heterogeneity of the common cyt c folding intermediate; 2) Cyt c molecules primarily sample interchanging extended and compact conformations on the folding energy landscape, the relative populations of which depend on denaturation conditions. The experimental approach will be to employ optical studies of folding on the single-molecule (SM) level, which will allow analysis of conformations and dynamics of individual members of an ensemble of polypeptides. Proposed studies will involve both time-integrated and time resolved SM fluorescence resonance energy transfer measurements, whereby mutations to residues on the protein surface will allow for access to dynamics of protein structure. Cyt c offers a number of key advantages: 1) The covalently attached heme group provides an intrinsic acceptor in energy transfer experiments, thus simplifying sample preparation and minimizing perturbation of protein folding by derivatization. 2) Cyts c are highly soluble, stable, and robust proteins. 3) Cyts c fold simply (i.e. approximated by a two-state model), which is important, as complexity will arise as members of the ensemble are probed. 4) Cyt c folding is well characterized on the macroscopic level, but not yet characterized on the single-molecule level.

该项目包含一项综合研究和教育计划，涉及生物物理学研究方面的本科生和研究生的指导、教育和培训。该项目的目标是了解细胞色素c(Cytc)折叠的分子复杂性，为描述细胞色素c的能量格局提供至关重要的数据。测量将检验与理解蛋白质折叠相关的两个基本假设：1)氨基酸序列的螺旋倾向的程度和分布在决定常见的细胞色素c折叠中间体的稳定性、集中性和异质性方面起重要作用；2)细胞色素c分子主要在折叠能量图谱上采样互换的扩展和紧凑的构象，其相对密度取决于变性条件。实验方法将是在单分子(SM)水平上使用折叠的光学研究，这将允许分析多肽集合的单个成员的构象和动力学。拟议的研究将涉及时间积分和时间分辨SM荧光共振能量转移测量，借此蛋白质表面残基的突变将允许获得蛋白质结构的动力学。Cytc提供了许多关键的优点：1)在能量转移实验中，共价连接的血红素基团提供了一个内在的受体，从而简化了样品的制备，并通过衍生化将蛋白质折叠的扰动降至最低。2)Cytsc是一种高度可溶、稳定和强健的蛋白质。3)简单地(即，由两态模型近似)，这是重要的，因为随着对系综成员的探索，复杂性将会出现。4)细胞色素c折叠在宏观水平上已经得到了很好的表征，但在单分子水平上还没有得到很好的表征。