Kinetics of Early Events in Protein Folding

蛋白质折叠早期事件的动力学

• 批准号: 7922834
• 负责人: HEINRICH RODER
• 金额: $ 9.69万
• 依托单位: RESEARCH INST OF FOX CHASE CAN CTR
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7922834
• 关键词: Acids; Address; Binding; Biological; Blood capillaries; Calculi; Chemicals; Complement; Coupled; Coupling; Data; Detection; Devices; Dimensions; Disease; Dyes; Elements; Energy Transfer; Engineering; Equilibrium; Equus caballus; Event; Exhibits; Fluorescence; Fluorescence Resonance Energy Transfer; Heme; Hydrogen Bonding; In Vitro; Individual; Kinetics; Label; Link; Measurement; Measures; Methods; Micrococcal Nuclease; Modeling; Modification; Molecular Conformation; Monitor; Mutation; Nature; Optics; Physiologic pulse; Play; Population; Positioning Attribute; Property; Protein Engineering; Proteins; Reaction; Relative (related person); Residual state; Roentgen Rays; Role; Staging; Structure; Techniques; Testing; Thermodynamics; Time; Tryptophan; Urea; Variant; base; capillary; cytochrome c; design; infrared spectroscopy; insight; light scattering; millisecond; mutant; numb protein; protein folding; protein structure prediction; rapid technique; research study; trafficking

DESCRIPTION (provided by applicant): This project continues to focus on understanding the role of early structural events in the transition from the unfolded to the native state of a protein, which is one of the most challenging and critical aspects of the protein folding problem. Rapid accumulation of partially folded intermediates may be important for directing the protein toward its unique native conformation. Despite intense study, important questions remain to be answered concerning the nature and origin of the barriers and the structural properties of the intermediates encountered during early stages of folding. Are compact states the result of a non- specific chain collapse or more specific folding events? What is the relative importance of local vs. long- range interactions? Do intermediates contain native-like tertiary interactions? For many proteins, intermediates are populated even at equilibrium, which raises further questions concerning the origin of structural co-operativity, the relationship between kinetic and thermodynamic intermediates, and any residual interactions remaining in the denatured state. These fundamental questions are addressed by coupling advanced mixing techniques for rapid initiation of folding reactions with a variety of detection methods, including intrinsic and extrinsic fluorescence probes and H/D exchange labeling experiments with NMR detection. These techniques, in conjunction with protein engineering and chemical modification methods for introducing spectroscopic marker, will be used to gain detailed insight into the folding mechanism of model proteins, including staphylococcal nuclease and horse cytochrome c. The Specific Aims are: (1) to monitor the formation of long-range and specific tertiary contacts during folding of these proteins by coupling fluorescence labeling and microsecond mixing techniques; (2) to observe formation of hydrogen-bonded structure on the microsecond time scale by H/D exchange and NMR; (3) to elucidate the structure of equilibrium intermediates; (4) to study the conformational properties of denatured protein states by fluorescence and hydrodynamic methods. Insight into the structural, thermodynamic and kinetic properties of protein folding intermediates is critical for understanding and treating a wide range of diseases that can be linked to aggregation of partially denatured or misfolded forms of proteins. Issues related to protein stability and folding also play a central role in understanding the biological consequences of mutations, and in de novo protein design. Studies of protein folding in vitro further provide the necessary framework for protein structure prediction, and for understanding cellular protein folding, trafficking and degradation.

描述（由申请人提供）：该项目继续专注于理解早期结构事件在从展开的蛋白质的本地状态过渡中的作用，这是蛋白质折叠问题的最具挑战性和最关键的方面之一。部分折叠中间体的快速积累对于将蛋白质引导到其独特的天然构象可能很重要。尽管进行了激烈的研究，但关于障碍的性质和起源以及在折叠早期阶段遇到的中间体的结构特性的重要问题仍有待解决。紧凑的状态是非特异性链塌陷还是更特定的折叠事件的结果？本地与长距离互动的相对重要性是什么？中间体是否包含类似天然的三级相互作用？对于许多蛋白质，中间体即使在平衡处也被人群，这提出了有关结构合作的起源，动力学和热力学中间体之间的关系以及在变性状态中仍然存在的任何残留相互作用的问题。这些基本问题是通过将高级混合技术耦合，以通过多种检测方法快速启动折叠反应，包括内在和外部荧光探针以及NMR检测的H/D交换标记实验。这些技术与蛋白质工程和用于引入光谱标记的化学修饰方法结合使用，将用于获得对模型蛋白质的折叠机制的详细见解，包括葡萄球菌核酸酶和马匹细胞色素c。具体目的是：（1）通过耦合荧光标签和微秒混合技术来监测这些蛋白质折叠过程中远程和特定三级接触的形成； （2）通过H/D交换和NMR观察在微秒时间尺度上形成氢键结构； （3）阐明平衡中间体的结构； （4）通过荧光和流体动力学方法研究变性蛋白态的构象性能。洞悉蛋白质折叠中间体的结构，热力学和动力学特性对于理解和治疗可与部分变性或错误折叠形式的蛋白质的聚集有关的广泛疾病至关重要。与蛋白质稳定性和折叠有关的问题在理解突变的生物学后果以及从头蛋白质设计中也起着核心作用。体外蛋白质折叠的研究进一步为蛋白质结构预测提供了必要的框架，并了解细胞蛋白质折叠，运输和降解。