MECHANISMS OF PROTEIN UNFOLDING REACTIONS

蛋白质解折叠反应的机制

• 批准号: 6309158
• 负责人: ROBERT L BALDWIN
• 金额: $ 0.75万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-15 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6309158
• 关键词: biomaterials; biomedical resource; enzymes; nuclear magnetic resonance spectroscopy; proteins

How the polypeptide chain of a protein adopts its unique and biologically active three-dimensional structure is one of the most fundamental question in biology. To learn about the mechanism of protein folding, it is important to characterize the physical properties of intermediates in this reaction and to identify specific interactions that fold or unfold proteins. Specifically we propose heteronuclear NMR experiments to study the detailed mechanism of the acid-induced unfolding of apomyoglobin that proceeds from a "native" state N at pH 6 through partially unfolded intermediates I to the unfolded state U at pH 2. Protonation of histidine H24 and H119 has been suggested to trigger the N to I transition by breaking a specific hydrogen bond between H24 and H119 side chains. 1H-15N HMBC experiments will be used to follow the protonation and tautomeric state of all histidine residues at various pH values. Aspartic acid residues that may be responsible for the I to U transition will be identified by modified 1H-13C CT-HCACO experiments at various pH values using apomyoglobin specifically labeled with 13C aspartic acid. Recent 1H NMR experiments suggested the existence of an intermediate in the unfolding reaction of ribonuclease A. Further characterization of this unfolding intermediates would provide valuable information about the transition state of the reaction and therefore about the mechanism of unfolding. We propose to use 19F NMR in a real-time NMR unfolding experiment to test whether this intermediate has the properties of a dry molten globule, namely, freely rotating side chains in an unhydrated protein interior.

蛋白质的多肽链如何采用其独特的和 具有生物活性的三维结构是最具生物活性的 生物学中的基本问题。要了解的机制 蛋白质折叠，重要的是要表征物理 该反应中中间体的性质，并确定特定的 折叠或展开蛋白质的相互作用。具体来说，我们建议 异核核磁共振实验研究其详细作用机理 酸诱导脱脂蛋白去折叠，脱脂蛋白来源于“天然的” 状态N在pH 6通过部分展开的中间体I到 组氨酸H24和H119的质子化 建议通过打破特定的 H24和H119侧链之间的氢键。1H-15N HMBC 实验将用来跟踪质子化和互变异构 所有组氨酸残基在不同pH值下的状态。天冬氨酸 可能导致I到U转变的残基将是 不同pH条件下改进的~1H-13C CT-HCACO实验鉴定 用13C天冬氨酸标记的去肌红蛋白测定值。 最近的~1H核磁共振实验表明存在一种中间体 在核糖核酸酶A的去折叠反应中进一步表征 将提供有价值的信息 关于反应的过渡态，因此关于 展开机制。我们建议在实时核磁共振中使用~(19)F核磁共振 展开实验，以测试该中间体是否具有 干熔球的性质，即自由旋转的侧面 在未加水的蛋白质内部形成链条。