BIOPHYSICAL STUDIES OF FOLDING MUTANTS OF STAPH NUCLEASE

葡萄球菌核酸酶折叠突变体的生物物理学研究

• 批准号: 2701514
• 负责人: DAVID Robert SHORTLE
• 金额: $ 29.9万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-05-01 至 1999-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2701514
• 关键词: Staphylococcus; acidity /alkalinity; biophysics; chemical kinetics; chemical stability; computer program /software; conformation; dipole moment; enzyme structure; fluorescence spectrometry; gene mutation; hydropathy; mathematical model; mutant; nuclear magnetic resonance spectroscopy; nuclease; peptide chemical synthesis; protein denaturation; protein folding; protein sequence; site directed mutagenesis; structural biology; synthetic peptide; thermodynamics

The long term objective of the research described in this application continues to be the development of a quantitative model that describes how the 149 amino acids of staphylococcal nuclease determine the structure of its native state, its stability, and its folding pathway. To achieve this objective, an extensive NMR analysis will be made of the residual structure which persists in partially folded forms of nuclease. Preliminary work indicates that the amount and nature of this residual structure varies with the perturbation that causes break down of the folded, native state. Initial characterization of a low density denatured state, a large fragment designated delta131delta, will be systematically extended by fully assigning all of the side chain 1H and 13C resonances. An exhaustive search for side chain/side chain NOE's will involve the development of new pulse sequences that filter out intraresidue NOE's through the incorporation of specific 13C labelled amino acid residues and that transfer an NOE signal from side chain protons to the much more disperse backbone protons (H-alpha and H/N for detection. This structural analysis will be pursued to the highest level of resolution attainable. In order to quantitate the relative stability of different structural elements in delta131delta, NMR analysis will be undertaken in the presence of denaturants (e.g., urea) and stabilizers (e.g., glycerol). The dependence of these structural elements on other regions of the polypeptide chain will be investigated using both large peptides labeled with 15N/13C generated by chemical cleavage at uniquely introduced cysteine residues plus chemically synthesized small peptides. Refinement of a structural model of a highly compact "quasi-native" form of nuclease will be pursued to high resolution, and the residual structure of a number of partially folded forms of nuclease induced by single substitution and insertion mutations will be analyzed and compared to the results obtained with delta131delta. The ultimate goal of this work is to construct an "equilibrium folding pathway" for nuclease, one that represents the stabilities and the hierarchical interdependencies of the major chain/chain interactions. In effect, the "free energy distance" between the denatured and native states becomes the axis along which structure develops, taking the place of time as the variable in the more conventional kinetic folding approach. And finally, empirical equations for calculating the stability effects of alanine and glycine substitutions in staph nuclease will be developed, refined, and tested by building upon the extensive mutant database already in hand and by employing mathematical techniques such as statistical factor analysis, neural networks and fuzzy logic.

本申请中描述的研究的长期目标 继续发展一种量化模型，描述 葡萄球菌核酸酶的149个氨基酸如何决定 它的天然状态的结构、它的稳定性和它的折叠途径。 为了实现这一目标，将进行广泛的核磁共振分析 以部分折叠的核酸酶形式存在的残存结构。 初步工作表明，残留物的数量和性质 结构随引起分解的扰动而变化 折叠的，原生的状态。低密度的初始表征 变性状态，一个名为delta131 Delta的大片段，将是 通过完全分配所有侧链1H和 13C共振。对侧链/侧链NOE的穷尽搜索 将涉及到新脉冲序列的开发，这些脉冲序列可以过滤掉 通过掺入特定的13C标记的残基NOE 氨基酸残基，从侧链传递NOE信号 质子到更分散的主干质子(H-α和H/N对于 侦测。这种结构分析将达到最高水平 可达到的解决方案。为了量化相对稳定性 对于Delta131Delta的不同结构元素，核磁共振分析将是 在存在变性剂(如尿素)和稳定剂的情况下进行 (例如，甘油)。这些结构元素对其他元素的依赖 多肽链的区域将使用两个大的 化学裂解产生的15N/13C标记的多肽 引入半胱氨酸残基和化学合成的小肽。 高度紧凑的“准自然”形式的结构模型的改进 核酸酶将被追求到高分辨率，残留物 几种部分折叠形式的核酸酶的结构 单替换和插入突变将被分析和比较 与用Delta131Delta得到的结果相一致。这样做的最终目的是 工作是为核酸酶构建一条“平衡折叠路径”。 表示稳定性和层次结构相互依赖关系的 主要的链/链相互作用。实际上，“自由能” 变性状态和自然状态之间的距离成为沿 哪种结构发展起来，以时间的位置为变量 更传统的动力学折叠方法。最后，经验性的 丙氨酸和甘氨酸稳定性影响的计算公式 葡萄球菌核酸酶的替代将被开发、提炼和测试。 通过建立在已有的广泛的突变数据库上，并通过 使用诸如统计因子分析等数学技术， 神经网络和模糊逻辑。