BIOPHYSICAL STUDIES OF FOLDING MUTANTS OF STAPH NUCLEASE

本申请中描述的研究的长期目标是 开发了一个定量模型来描述149个氨基酸如何 葡萄球菌核酸酶决定了天然状态的结构，其 稳定性，及其折叠途径。为了实现这一目标，广泛的 将利用基因工程方法系统地改变 这种小蛋白质的氨基酸序列有多种方式。1)每个 氨基酸残基将被突变为丙氨酸和甘氨酸以去除 野生型侧链。2)单一的丙氨酸和甘氨酸残基 插入到多对野生型残基之间以改变 链段之间的间距。3)20种氨基酸中的许多将是 在几个选定的位置替换，以检查不同 侧链。4)随机诱导的突变，降低了稳定性 核酸酶的可逆变性将使用简单的平板进行恢复 化验。为了量化这些序列修改对 结构和折叠，各种生物物理方法将被用在 高纯度突变蛋白的鉴定。作为第一步， 变性过程中的自由能变化和变化率 关于变性剂的浓度将通过监测确定 用本征荧光和循环平衡展开反应 二向色性。然后将在这两个值和一个 中描述残留物的局部环境的参数数量 原生状态。对于不寻常的突变体，如稳定插入， 结构后果将通过x射线结晶学来确定。 方法与其他实验室合作。看起来像是 改变残存结构的变性状态将被重组为 核酸酶的大片段(作为变性状态的模型) 并用圆二色谱和凝胶对其剩余结构进行了定量 过滤。检测突变对Full变性状态的影响 长度蛋白质，荧光能量转移技术将是 使用。一种假定的折叠中间状态的详细研究 将继续以疏水相互作用为主，包括 高分辨质子核磁共振结构研究。该系统的运动学分析 将启动折叠和展开的速度。以及它们的图案 许多小蛋白中的疏水接触，包括葡萄球菌 核酸酶突变体，将用数学工具来表征 图论。

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.