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

细胞色素 c 折叠中屏障和中间体的结构和动力学表征

• 批准号: 0079148
• 负责人: Heinrich Roder
• 金额: $ 36万
• 依托单位: Institute For Cancer Research
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2004-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0079148&HistoricalAwards=false
• 关键词: Structural; Kinetic; Characterization; Barriers; Intermediates

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.

这个项目的目标是提供对控制球状蛋白自发折叠的结构和能量因素的透彻理解。将结合位点诱变、超快速混合技术、氢交换标记和核磁共振技术，研究一个小的单域蛋白马细胞色素c的折叠和展开的动力学机制。为了确定在折叠过程中稳定早期中间体和过渡态集合的关键相互作用，整个a-螺旋核心的疏水残基将被改变，并且由此产生的平衡和动力学参数的变化将被测量为变性剂浓度的函数。第二系列细胞色素c变异体将被准备用来测试极性三级相互作用在折叠后期形成的假设，并有助于天然结构的协同性。对每个突变体的动力学结果进行定量分析，将提供折叠不同阶段中单个残基参与的信息，包括在微秒时间尺度上链的初始崩溃，随后获得部分结构状态和本地结构的限速形成。通过氢交换标记和核磁共振方法将获得早期折叠中间体中氢键结构的补充信息。将通过结合灵敏的爆发相标记方案和新型毛细管混合装置来测量亚毫秒时间尺度上单个酰胺质子对H/D交换的保护。由此获得的对中间体和过渡态集成体的结构洞察力将确定涉及其稳定性的关键相互作用，这是理解该蛋白质折叠和稳定性的序列决定因素的重要一步。这些发现将阐明蛋白质折叠的基本问题，包括a)在多肽链初始崩溃过程中遇到的动力学屏障的性质和起源，b)中间产物在指导折叠中的作用，c)限速能垒的性质以及d)折叠/展开转变中的协同性。突变分析将为以下假设提供关键检验：球形蛋白核心的保守疏水接触是在折叠早期形成的，对有效折叠很重要，而特定的侧链填充和极性三级相互作用是在折叠后期建立的，对天然结构的刚性和协同性很重要。研究结果将为蛋白质折叠、折叠识别、结构预测和蛋白质从头设计的理论和计算模型的测试提供坚实的实验基础。