MECHANISMS OF PROTEIN UNFOLDING REACTIONS

蛋白质解折叠反应的机制

• 批准号: 6281522
• 负责人: ROBERT L BALDWIN
• 金额: $ 0.6万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-01 至 1999-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6281522
• 关键词: biomaterials; biomedical resource; enzymes; nuclear magnetic resonance spectroscopy; proteins; 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和 生物活性的三维结构是最重要的结构之一 生物学的基本问题。 了解 蛋白质折叠，表征物理很重要 中间体在此反应中的特性，并确定特定 折叠或展开蛋白质的相互作用。 具体来说，我们建议 异核NMR实验研究了 酸诱导的apomyoglobin的展开是从“天然”进行的 通过部分展开的中间体i到6处的状态n pH 2的展开状态u。组氨酸H24和H119的质子化具有 建议通过打破特定的特定 H24和H119侧链之间的氢键。 1H-15N HMBC 实验将用于遵循质子化和互变异层。 在各种pH值下所有组氨酸残基的状态。 天冬氨酸 可能导致i向U转变的残留物将是 通过修改的1H-13C CT-HCACO实验在各种pH 使用阿疗蛋白特异性标记为13C天冬氨酸的值。 最近的1H NMR实验表明存在中间体 在核糖核酸酶的展开反应中。 其中展开的中间体将提供有价值的信息 关于反应的过渡状态，因此 展开的机制。 我们建议在实时NMR中使用19F NMR 展开实验以测试该中间体是否具有 干熔球的特性，即自由旋转的侧面 未氢氢蛋白质内部的链条。