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: 2177317
• 负责人: DAVID Robert SHORTLE
• 金额: $ 29.34万
• 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 1995-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2177317
• 关键词: Staphylococcus; X ray crystallography; enzyme structure; fluorescence spectrometry; genetic manipulation; hydropathy; mathematical model; molecular cloning; mutant; nuclear magnetic resonance spectroscopy; nuclease; protein denaturation; protein folding; protein sequence; transposon /insertion element

The long term objective of the research described in this application is the development of a quantitative model to describe how the 149 amino acids of staphylococcal nuclease determine the structure of the native state, its stability, and its folding pathway. To achieve this objective, extensive use will be made of genetic engineering methods to systematically alter this small protein's amino acid sequence in a variety of ways. 1) Each amino acid residue will be mutated to both alanine and glycine to remove the wild-type side chain. 2) Single alanine and glycine residues will be inserted between a number of pairs of wild-type residues to alter the spacing between chain segments. 3) Many of the 20 amino acids will be substituted at several select positions to examine the effect of different side chains. 4) Randomly induced mutations that lower the stability of nuclease to reversible denaturation will be recovered using a simple plate assay. To quantitate the effects of these sequence modifications on structure and folding, a variety of biophysical methods will be used in the characterization of highly purified mutant proteins. As a first step, the free energy change deltaG on denaturation and the rate of change of delta G with respect to denaturant concentration will be determined by monitoring the equilibrium unfolding reaction via intrinsic fluorescence and circular dichroism. Correlations will then be sought between these two values and a number of parameters that describe a residue's local environment in the native state. For unusual mutants such as stable insertions, the structural consequences will be identified by x-ray crystallographic methods in collaboration with other laboratories. Mutants that appear to alter the residual structure of the denatured state will be recombined into large fragments of nuclease ( which serve as models of the denatured state) and their residual structure quantitated by circular dichroism and gel filtration. To examine mutant effects on the denatured state of full length protein, the techniques of fluorescence energy transfer will be used. A detailed study of a presumptive intermediated state of folding dominated by hydrophobic interactions will be continued, including structural studies by high resolution proton NMR. Kinetic analysis of the rates of folding and unfolding will be initiated. And the patterns of hydrophobic contacts in a number of small proteins, including staph nuclease mutants, will be characterized with the mathematical tools of Graph Theory.

本应用程序中描述的研究的长期目标是 定量模型的开发，以描述149个氨基酸如何 葡萄球菌核酸酶确定天然状态的结构，其 稳定性及其折叠路径。 实现这一目标 将使用基因工程方法进行系统改变 这种小蛋白质的氨基酸序列以多种方式。 1）每个 氨基酸残留物将突变为丙氨酸和甘氨酸以去除 野生型侧链。 2）单丙氨酸和甘氨酸残基将是 插入多对野生型残基之间以更改 链段之间的间距。 3）20个氨基酸中的许多将是 在几个选择的位置上取代以检查不同的效果 侧链。 4）随机诱导的突变，以降低 将使用简单的板恢复可逆变性的核酸酶 测定。 量化这些序列修改对 结构和折叠，将在 高度纯化的突变蛋白的表征。 作为第一步， 自由能量变性三角洲关于变性和增量的变化率 关于变性浓度，将通过监视确定 通过内在荧光和圆形的平衡展开反应 二分性能。 然后，将在这两个值和a之间寻求相关性 描述残基的本地环境的参数数量 本地国家。 对于异常突变体，例如稳定插入， 结构后果将通过X射线晶体学标识 与其他实验室合作的方法。 突变体似乎 更改变性状态的残留结构将被重新组合到 核酸酶的大片段（作为变性状态的模型） 及其残留结构通过圆形二色性和凝胶定量 过滤。 检查突变体对完整状态的变性状态 长度蛋白质，荧光能量转移的技术将是 用过的。 对推定的中间折叠状态的详细研究 以疏水相互作用为主的 高分辨率质子NMR的结构研究。 动力学分析 折叠率和展开率将开始。 和模式 许多小蛋白质中的疏水接触，包括葡萄球菌 核酸酶突变体将以数学工具为特征 图理论。