BIOPHYSICAL STUDIES OF FOLDING MUTANTS OF STAPH NUCLEASE

本申请中描述的研究的长期目标是 开发一个定量模型来描述149种氨基酸 的葡萄球菌核酸酶决定的结构的天然状态，其 稳定性及其折叠途径。 为了实现这一目标， 将利用基因工程方法系统地改变 这个小蛋白的氨基酸序列。 1)每个 氨基酸残基将突变为丙氨酸和甘氨酸 野生型侧链。 2)单个丙氨酸和甘氨酸残基将是 插入到许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 在几个选定的位置取代，以检查不同的效果， 侧链。 4)随机诱导的突变降低了 将使用简单平板回收可逆变性的核酸酶 比色法 为了定量这些序列修饰对 结构和折叠，各种生物物理方法将用于 高度纯化的突变蛋白的表征。 作为第一步 变性时的自由能变化Δ G和Δ G的变化率 将通过监测来确定变性剂浓度 通过内源荧光和环状构象的平衡去折叠反应 二色性 然后将在这两个值之间寻找相关性， 描述残留物在环境中的局部环境的参数的数量 本土国家。 对于不寻常的突变体，如稳定插入， 将通过X射线晶体学鉴定结构结果 与其他实验室合作。 突变体似乎 改变变性状态的残余结构将重组成 核酸酶的大片段（作为变性状态的模型） 并通过圆二色谱和凝胶色谱定量了它们的残余结构 过滤 为了检查突变体对完全变性状态的影响 长度的蛋白质，荧光能量转移技术将是 采用 一个假定的褶皱中间状态的详细研究 由疏水相互作用主导的研究将继续进行，包括 通过高分辨率质子NMR进行结构研究。 动力学分析 将启动折叠和展开的速率。 而这些模式 疏水接触在一些小的蛋白质，包括葡萄球菌 核酸酶突变体，将与数学工具， 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.