Single-molecule fluorescence studies of protein folding

蛋白质折叠的单分子荧光研究

• 批准号: 7587261
• 负责人: Gilad Haran
• 金额: $ 24.85万
• 依托单位: WEIZMANN INSTITUTE OF SCIENCE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7587261
• 关键词: ALPP; Address; Alzheimer' s Disease; Amino Acids; Behavior; Cereals; Circular Dichroism; Collaborations; Consensus; Cysteine; Dependence; Diffuse; Diffusion; Disease; Dyes; Enzymes; Equilibrium; Family; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescence Spectroscopy; Free Energy; Generations; Goals; Heterogeneity; Individual; Kinetics; Label; Learning; Ligation; Lipids; Maps; Measurement; Measures; Methodology; Methods; Microscopic; Modeling; Molecular Conformation; Mutation; Non-Insulin-Dependent Diabetes Mellitus; Pathway interactions; Physiology; Population; Positioning Attribute; Process; Protein Dynamics; Proteins; Reporting; Research; Research Personnel; Resolution; Site; Spectrum Analysis; Structure; Surface; System; Techniques; Tertiary Protein Structure; Thermodynamics; Time; Tryptophan; Universities; Vesicle; adenylate kinase; base; programs; protein aggregate; protein folding; research study; single molecule; three dimensional structure; tool

DESCRIPTION (provided by applicant): Understanding pathways of protein folding and unfolding, and mapping partially folded intermediate states, are of utmost importance for our ability to decipher the physiology of misfolding diseases. The generation of maps of folding pathways on the energy landscapes of proteins is the long range goal of our research. Here we propose to address this goal by applying powerful single-molecule fluorescence methodology to study the folding of selected protein systems. We will use fluorescence resonance energy transfer (FRET) to follow conformational dynamics of protein molecules trapped within surface-tethered lipid vesicles, a unique method developed in our lab. Vesicles will be prepared under conditions where molecules have equal folding and unfolding rates, so that single- molecule trajectories will show frequent transitions between conformational states, including partially-folded intermediates. Analysis of trajectories will yield a wealth of information on folding pathways and rates of interconversion between folding intermediates. In addition, studies on freely-diffusing molecules will probe global structural changes of the proteins during folding. We will first study the 62 amino-acid protein L, known to be a two-state folder from bulk experiments. We will verify this observation on the single molecule level, as well as obtain structural information on the denatured state of the protein. We will also study a larger protein, adenylate kinase, which, as we already showed, possesses a very heterogeneous folding energy lansdcape. Intermediates observed during folding of this protein will be carefully mapped. A low-resolution structure of these intermdiates will be obtained by measuring several pairs of labeling sites. In a strong collaboration with Lynne Regan from Yale we will study single-molecule folding of the tetratricopeptide repeat proteins. This family of modular proteins, constructed of 34 amino-acid repeat units, has been suggested to obey a new folding paradigm, based on the classical Ising model, which predicts a large population of partially folded states near the transition midpoint. Single-molecule measurements will probe this intriguing assertion. This study will have impact on our understanding of misfolding diseases, like Alzheimer's and type II diabetes. Our experiments should help us identifying intermediate structures that act as 'weak points' during folding, leading to protein aggregates which are responsible for disease.

描述（由申请人提供）：了解蛋白质折叠和展开的途径，并绘制部分折叠的中间状态，对于我们破译错误折叠疾病的生理学至关重要。在蛋白质的能量版图上生成折叠路径图是我们研究的长期目标。在这里，我们建议通过应用强大的单分子荧光方法来研究选定蛋白质系统的折叠来解决这一目标。我们将使用荧光共振能量转移（FRET）来跟踪被困在表面系住的脂质囊泡内的蛋白质分子的构象动力学，这是我们实验室开发的一种独特方法。囊泡将在分子具有相同折叠和展开速率的条件下制备，因此单分子轨迹将在构象状态之间频繁转换，包括部分折叠的中间体。对轨迹的分析将产生关于折叠路径和折叠中间体之间相互转换率的丰富信息。此外，对自由扩散分子的研究将探索蛋白质在折叠过程中的整体结构变化。我们将首先研究62个氨基酸的蛋白质L，已知是一个双态文件夹从批量实验。我们将在单分子水平上验证这一观察结果，并获得关于蛋白质变性状态的结构信息。我们还将研究一个更大的蛋白质，腺苷酸激酶，正如我们已经展示的，它具有非常异质的折叠能量格局。在蛋白质折叠过程中观察到的中间产物将被仔细地绘制出来。这些中间体的低分辨率结构将通过测量几对标记位点得到。与耶鲁大学的Lynne Regan合作，我们将研究四肽重复蛋白的单分子折叠。这个由34个氨基酸重复单元组成的模块化蛋白家族已经被认为遵循一种新的折叠范式，基于经典的Ising模型，该模型预测在过渡中点附近存在大量部分折叠状态。单分子测量将探测这个有趣的断言。这项研究将影响我们对错误折叠疾病的理解，如阿尔茨海默氏症和II型糖尿病。我们的实验将帮助我们识别中间结构，这些中间结构在折叠过程中充当“弱点”，导致导致疾病的蛋白质聚集。