STRUCTURAL CHARACTERIZATION OF GLOBIN FOLDING PATHWAYS

球蛋白折叠通路的结构特征

• 批准号: 2684143
• 负责人: PETER Edwin WRIGHT
• 金额: $ 23.87万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-12-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2684143
• 关键词: biochemical evolution; chemical kinetics; chemical stability; circular dichroism; conformation; fluorescence spectrometry; hemoglobin; molecular cloning; myoglobin; nuclear magnetic resonance spectroscopy; polymerase chain reaction; protein denaturation; protein folding; protein purification; protein reconstitution; protein sequence; protein structure; site directed mutagenesis; stable isotope; stop flow technique; structural biology; tissue /cell culture

Understanding the fundamental molecular mechanisms by which proteins fold into the precise three-dimensional structures required for biological activity remains one of the most challenging problems in structural biology. It is generally accepted that all of the information required for correct folding is coded within the amino acid sequence; just how that code is translated into folding pathways and highly specific tertiary structure is not yet known. There is an urgent need for detailed information on the structures of folding intermediates at the level of individual amino acid residues, and a need for a deeper understanding of the factors that influence their rates of formation and stability. The overall objective of the proposed research is to address some of these outstanding issues through kinetic and equilibrium studies of intermediates formed on the folding pathway of apomyoglobin. Apomyoglobin is ideally suited for such studies because a stable molten globule intermediate that is formed early on the kinetic folding pathway can also be obtained under equilibrium conditions appropriate for high resolution NMR experiments. A combination of mutagenesis, stopped-flow kinetics measurements, hydrogen exchange pulse labeling, and state-of-the-art heteronuclear NMR experiments will be applied to investigate the kinetic folding pathway of apomyoglobin and characterize the structure of the stable molten globule folding intermediate. Mutations will be introduced at specific sites to probe the molecular interactions that stabilize the kinetic folding intermediates, and to investigate the effect of alterations in the intrinsic secondary structural propensities of the polypeptide on the rate of formation and stability of the molten globule intermediate. Direct heteronuclear NMR methods will be used to obtain critical information on the structure, dynamics, and state of hydration of the equilibrium molten globule intermediate of apomyoglobin at the level of single residues. This research program is expected to provide important new insights into the structure and stabilization of folding intermediates at an unprecedented level of detail, and will add significantly to the understanding of protein folding mechanisms. In addition, the kinetic folding pathway of an evolutionarily distant plant leghemoglobin will be characterized, along with the structure of a partly folded intermediate. These studies are expected to provide a particularly stringent test of the hypothesis that folding pathways are conserved in homologous proteins.

了解蛋白质折叠的基本分子机制 转化为生物学所需的精确三维结构 活动仍然是结构性最具挑战性的问题之一， 生物学一般认为，所有需要的信息， 正确的折叠是在氨基酸序列中编码的， 编码被翻译成折叠途径和高度特异性的三级结构 结构尚不清楚。迫切需要详细的 折叠中间体的结构信息， 单个氨基酸残基，以及需要更深入地了解 影响其形成速率和稳定性的因素。的 拟议研究的总体目标是解决其中的一些问题， 通过动力学和平衡研究， 在脱辅基肌红蛋白的折叠途径上形成的中间体。脱辅基肌红蛋白 非常适合这种研究，因为稳定的熔融球 在动力学折叠途径早期形成的中间体也可以 在适于高分辨率的平衡条件下获得 NMR实验。结合诱变，停流动力学 测量、氢交换脉冲标记和最新技术 将应用异相NMR实验来研究动力学 的折叠途径和表征的结构， 稳定的熔融球折叠中间体。突变将被引入 在特定的位点探测分子间的相互作用， 动力学折叠中间体，并研究影响 改变内在的二级结构倾向的 多肽对熔融球形成速率和稳定性的影响 中间体将使用直接杂原子NMR方法来获得 关于结构、动力学和水化状态的关键信息 去氧肌红蛋白的平衡熔融球中间体， 单一残留物。这项研究计划预计将提供重要的 对折叠中间体的结构和稳定性的新见解 以前所未有的细节水平，并将大大增加 了解蛋白质折叠机制。 此外，一个进化上遥远的 植物豆血红蛋白将被表征，沿着具有以下结构： 部分折叠的中间体。预计这些研究将提供一个 特别严格的假设，即折叠途径是 在同源蛋白质中是保守的。