BIOPHYSICAL STUDIES OF FOLDING MUTANTS OF STAPH NUCLEASE

本申请中描述的研究的长期目标 仍然是一个定量模型的发展， 葡萄球菌核酸酶的149个氨基酸是如何决定 其天然状态的结构、其稳定性和其折叠途径。 为了实现这一目标，将进行广泛的NMR分析， 以核酸酶的部分折叠形式存在的残余结构。 初步工作表明，这一残留物的数量和性质 结构随扰动而变化， 折叠的，原生状态。 低密度的初步表征 在变性状态下，一个命名为delta 131 delta的大片段将被 通过完全分配所有侧链1H和1H， 13 C共振。 对侧链/侧链NOE的详尽搜索 将涉及开发新的脉冲序列， 通过掺入特异性13 C标记的残基内NOE 氨基酸残基，并从侧链转移NOE信号 质子到更分散的主链质子（对于H-alpha和H/N， 侦测 这一结构分析将在最高级别进行 的分辨率。 为了定量相对稳定性 δ 131 δ中的不同结构元素，NMR分析将是 在变性剂存在下进行（例如，尿素）和稳定剂 (e.g.,甘油）。 这些结构要素对其他 多肽链的区域将使用两个大的 通过化学裂解产生的15 N/13 C标记肽， 引入半胱氨酸残基加上化学合成的小肽。 一个高度紧凑的“准原生”形式的结构模型的改进 核酸酶将被追求到高分辨率，和残留的 诱导的许多部分折叠形式的核酸酶的结构 将分析和比较单个置换和插入突变 与用delta 131 delta获得的结果一致。 这个项目的最终目标 工作是构建核酸酶的“平衡折叠途径”， 它代表稳定性和分层相互依赖性 链与链之间的相互作用。 实际上，“自由能” 变性状态和天然状态之间的“距离”成为沿着沿着的轴 这种结构的发展，以时间作为变量的地方， 更传统的动力学折叠方法。 最后，经验主义 计算丙氨酸和甘氨酸稳定性影响的方程 葡萄球菌核酸酶中的取代将被开发、改进和测试， 通过建立在已经掌握的广泛的突变体数据库上， 采用诸如统计因子分析的数学技术， 神经网络和模糊逻辑。

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.