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

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， 具有生物活性的三维结构是目前 生物学的基本问题。 为了了解 蛋白质折叠，重要的是要表征的物理 该反应中的中间体的性质，并确定具体的 折叠或展开蛋白质的相互作用。 具体来说，我们建议 通过异相NMR实验研究了 酸诱导的脱辅基肌红蛋白的解折叠， 在pH 6下通过部分未折叠的中间体I到 pH 2时的未折叠状态U。 组氨酸H24和H119的质子化具有 有人建议通过打破特定的 H24和H119侧链之间的氢键。 1H-15N HMBC 实验将被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 可能负责I到U转换的残基将是 在不同pH值下通过改良的1H-13 C CT-HCACO实验鉴别 使用13 C天冬氨酸特异性标记的脱辅基肌红蛋白的值。 最近的1H NMR实验表明存在中间体 在核糖核酸酶A的解折叠反应中。进一步表征 这些解折叠中间体的信息将提供有价值的信息 关于反应的过渡态， 展开的机制。 我们建议在实时NMR中使用19 F NMR 展开实验，以测试这种中间体是否具有 干燥熔融球的性质，即自由旋转侧 非水化蛋白质内部的链。