Structural and Kinetic Characterization of Barriers and Intermediates in Folding of Cytochrome c

RoderMCB 0079148这个项目的目标是提供对控制球状蛋白质自发折叠的结构和能量因素的彻底了解。通过结合定点突变和超快速混合技术、氢交换标记和核磁共振技术，将研究小的单域蛋白马细胞色素c的折叠和去折叠的动力学机制。为了确定在折叠过程中稳定早期中间体和过渡态系综所涉及的关键相互作用，将改变整个a-螺旋核心的疏水残基，并测量由此产生的平衡和动力学参数的变化作为变性剂浓度的函数。将准备第二系列细胞色素c变体，以检验极性三级相互作用在折叠后期形成并有助于天然结构的协作性的假设。对每个突变体的动力学结果的定量分析将提供有关单个残基在折叠的不同阶段参与的信息，包括微秒时间尺度上的链最初崩溃、随后部分结构状态的获得和天然结构的限速形成。通过氢交换标记和核磁共振方法可以获得关于早期折叠中间体氢键结构的补充信息。通过将敏感的突发相标记协议与新型毛细管混合设备相结合，将在亚毫秒时间尺度上测量单个酰胺质子对H/D交换的保护作用。由此获得的对中间体和过渡态系综的结构洞察将确定它们稳定所涉及的关键相互作用，这是理解该蛋白质折叠和稳定性的序列决定因素的关键一步。这些发现将阐明蛋白质折叠中的基本问题，包括a)在多肽链最初崩溃时遇到的动力学障碍的性质和来源，b)中间体在指导折叠中的作用，c)限速能量障碍的性质和d)折叠/去折叠转变中的协同性。突变分析将为以下假设提供关键检验：球状蛋白核心保守的疏水接触是在折叠早期形成的，对于有效的折叠是重要的，而特定的侧链堆积和极性三级相互作用是在折叠后期建立的，对于天然结构的刚性和协同性是重要的。这一结果将为验证蛋白质折叠、折叠识别、结构预测和从头设计的理论和计算模型提供坚实的实验基础。

RoderMCB 0079148The goal of this project is to provide a thorough understanding of the structural and energetic factors that govern the spontaneous folding of globular proteins. The kinetic mechanism of folding and unfolding of a small single-domain protein, horse cytochrome c, will be investigated by combining site-directed mutagenesis with ultra-fast mixing techniques, hydrogen exchange labeling and NMR. In order to identify critical interactions involved in stabilizing early intermediates and the transition state ensembles during folding, hydrophobic residues throughout the a-helical core will be altered, and the resulting changes in equilibrium and kinetic parameters will be measured as a function of denaturant concentration. A second series of cytochrome c variants will be prepared to test the hypothesis that polar tertiary interactions are formed late in folding and contribute to the cooperativity of the native structure. Quantitative analysis of the kinetic results for each mutant will provide information on the involvement of individual residues at various stages of folding, including the initial collapse of the chain on the microsecond time scale, the subsequent acquisition of partially structured states and the rate-limiting formation of the native structure. Complementary information on H-bonded structure in early folding intermediates will be obtained by hydrogen exchange labeling and NMR methods. Protection of individual amide protons against H/D exchange on the submillisecond time scale will be measured by combining a sensitive burst-phase labeling protocol with a novel capillary mixing device.The structural insight into intermediates and transition state ensembles thus obtained will identify key interactions involved in their stabilization, which is an essential step toward understanding the sequence determinants for folding and stability of this protein. The findings will elucidate fundamental issues in protein folding, including a) the properties and origin of the kinetic barrier encountered during the initial collapse of the polypeptide chain, b) the role of intermediates in directing folding, c) the nature of the rate-limiting energy barrier and d) the cooperativity in folding/unfolding transitions. The mutational analysis will provide a critical test for the hypothesis that conserved hydrophobic contacts in the core of globular proteins are formed early in folding and are important for efficient folding, while specific side chain packing and polar tertiary interactions are established late in folding and are important for the rigidity and cooperativity of the native structure. The results will provide a firm experimental basis for testing theoretical and computational models of protein folding, fold recognition, structure prediction and de novo protein design.