Kinetics of Early Events in Protein Folding

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蛋白质从未展开状态过渡到天然状态中的作用，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将蛋白质导向其独特的天然构象可能是重要的。尽管进行了大量的研究，但关于屏障的性质和起源以及在褶皱早期阶段遇到的中间体的结构性质等重要问题仍有待回答。紧化状态是由非特定链折叠或更特定的折叠事件引起的吗？本地与远程相互作用的相对重要性是什么？中间体是否包含原生的三级相互作用？对于许多蛋白质来说，中间体甚至在平衡状态下也被填充，这进一步提出了关于结构协同性的起源、动力学和热力学中间体之间的关系以及变性状态下剩余的相互作用的问题。这些基本问题是通过耦合先进的混合技术快速启动折叠反应与各种检测方法，包括内在和外在荧光探针和氢/D交换标记实验与核磁共振检测解决。这些技术，结合蛋白质工程和化学修饰方法引入光谱标记，将用于详细了解模型蛋白的折叠机制，包括葡萄球菌核酸酶和马细胞色素c。具体目的是：(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远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)利用荧光和流体动力学方法研究变性蛋白态的构象性质。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广泛疾病至关重要。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和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和理解细胞蛋白质折叠、转运和降解提供了必要的框架。

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.