HIERARCHY OF PROTEIN FOLDING AND STABILITY

结构生物学中主要的悬而未决的问题是蛋白质 折叠问题。 最后三强的获得规则- 大多数蛋白质的三维结构都编码在信息中， 然而，氨基酸的一级序列， 这些规则是缺乏的。 蛋白质折叠的一种流行模型是 层次模型，其中三级结构的形成， 蛋白质是通过二级结构单元的组装实现的， 包括α螺旋和β折叠的结构。 这种模式虽然 是吸引人的简单，一个直接的测试模型还没有 演示。 对模型肽中α-螺旋形成的研究提供了一种新的方法， 关于特定侧链贡献的丰富信息 结构形成的能量学的相互作用和 氨基酸的内在螺旋形成趋势。 的 这一信息对蛋白质稳定性，甚至对螺旋的适用性 在天然蛋白质衍生的肽中， 并且还没有执行规则的直接测试。 的可用性 两个相关的模型蛋白质系统，再加上研究孤立的 这些蛋白质的螺旋肽，将提供第一个直接测试 蛋白质稳定性的层次模型。 来自细菌的HPr蛋白是这些研究的理想载体， 来自大肠杆菌和枯草芽孢杆菌的蛋白质是 数量充足，可用于生物物理分析。 这两 蛋白质采用相同的三维结构， 34%序列同一性（86个残基中的30个）。 因此，HPr系统 提供了一个独特的机会来测试两个层次模型 不同的蛋白质，并比较结构的“上下文依赖性” 阵 代表这些蛋白质螺旋部分的肽 在水中作为分离的肽表现出大量的螺旋形成。 溶剂暴露，螺旋稳定的相互作用将在 两个相关的蛋白质，并与分离的肽的结果进行比较 为了得到蛋白质能量学的完整特征， 肽稳定性 HPr蛋白的其他片段将用于 解决二级结构形成在蛋白质稳定性中的作用 并量化特定物质之间相互作用的能量学 二级结构的元素。 30个相同的角色中的许多角色 两种蛋白质之间的残基在定义HPr折叠中将是 通过定向诱变研究确定。 长期目标是 以确定每个氨基酸残基对稳定性的贡献 和HPr的结构。 突变的结构性后果， 完整的蛋白质和肽片段都将通过NMR测定 光谱与氢交换相结合， 局部和全局构象突变的结构效应 稳定 到目前为止，没有一个单一的系统提供机会， 将分离肽的结构形成结果与 完整的蛋白质，并确定相同的突变的影响， 两个相关的蛋白质系统。

The major outstanding question in structural biology is the protein folding problem. Rules for the acquisition of the final three- dimensional structure of most proteins are encoded in the information in the primary sequence of amino acids, however, a precise description of these rules is lacking. One popular model for protein folding is the hierarchical model in which the formation of the tertiary structure of a protein is realized through the packing of elements of secondary structure including alpha-helices and beta-sheets. Although this model is appealing in its simplicity, a direct test of the model has not been demonstrated. The study of alpha-helix formation in model peptides has provided a wealth of information on the contributions of specific side-chain interactions to the energetics of structure formation and a measure of the intrinsic helix-forming tendencies of the amino acids. The applicability of this information to protein stability, or even to helix formation in peptides derived from native proteins, has only been assumed and a direct test of the rules has not been performed. The availability of two related model protein systems, coupled with studies on isolated helical peptides from those proteins, will provide the first direct test of the hierarchical model for protein stability. The HPr protein from bacteria is an ideal vehicle for those studies since the proteins from both Escherichia coli and Bacillus subtilis are available in sufficient quantities for biophysical analysis. These two proteins adopt the same three-dimensional structure despite having only 34% sequence identity (30 of 86 residues). Therefore, the HPr systems provide a unique opportunity to test the hierarchical model in two different proteins and to compare the "context-dependence" of structure formation. Peptides representing the helical portions of these proteins exhibit substantial helix formation as isolated peptides in water. Solvent-exposed, helix-stabilizing interactions will be studied in the two related proteins and compared with results from the isolated peptides to arrive at a complete characterization of the energetics of protein and peptide stability. Other fragments of the HPr proteins will be used to address the role of secondary structure formation in protein stability and to quantify the energetics of the interactions between specific elements of secondary structure. The role of many of the 30 identical residues between the two proteins in defining the HPr fold will be determined through directed mutagenesis studies. The long-term goal is to define the contribution of each amino acid residue to the stability and structure of HPr. The structural ramifications of the mutations, in both the intact proteins and peptide fragments, will be determine by NMR spectroscopy in combination with hydrogen exchange to correlate the structural effects of mutation with the local and global conformational stability. To date, no single system has provided the opportunity to compare the results from structure formation in isolated peptides with intact proteins and to determine the effects of identical mutations in two related protein systems.