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）：该项目继续致力于了解早期结构事件在蛋白质从未折叠状态向天然状态转变中的作用，这是蛋白质折叠问题中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引导蛋白质走向其独特的天然构象可能很重要。尽管进行了大量研究，但有关屏障的性质和起源以及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回答。紧凑状态是非特定链折叠或更特定折叠事件的结果吗？局部交互与远程交互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？对于许多蛋白质来说，即使在平衡状态下，中间体也会大量存在，这引发了有关结构协同性的起源、动力学和热力学中间体之间的关系以及处于变性状态的任何残余相互作用的进一步问题。这些基本问题可以通过将快速启动折叠反应的先进混合技术与各种检测方法相结合来解决，包括内在和外在荧光探针以及带有 NMR 检测的 H/D 交换标记实验。这些技术与引入光谱标记的蛋白质工程和化学修饰方法相结合，将用于详细了解模型蛋白质的折叠机制，包括葡萄球菌核酸酶和马细胞色素c。具体目标是：（1）通过耦合荧光标记和微秒混合技术来监测这些蛋白质折叠过程中长程和特定三级接触的形成； (2)通过H/D交换和NMR在微秒时间尺度上观察氢键结构的形成； (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质状态的构象特性。深入了解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与部分变性或错误折叠形式的蛋白质聚集有关的多种疾病至关重要。与蛋白质稳定性和折叠相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发挥着核心作用。体外蛋白质折叠研究进一步为蛋白质结构预测以及理解细胞蛋白质折叠、运输和降解提供了必要的框架。