HIERARCHY OF PROTEIN FOLDING AND STABILITY

结构生物学的主要突出问题是蛋白质。 折叠问题。关于收购最后三名的规则- 大多数蛋白质的空间结构都编码在 然而，氨基酸的主要序列，准确地描述了 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 蛋白质是通过次生元素的包装来实现的 结构包括α-螺旋和β-折叠。虽然这种模式 它的简单性很吸引人，但对该模型的直接测试还没有 演示了。 对模型多肽中α螺旋形成的研究提供了一种 关于特定侧链的贡献的丰富信息 与结构形成能量学的相互作用和一种测量 氨基酸的内在螺旋形成趋势。这个 这种信息对蛋白质稳定性的适用性，甚至对螺旋的适用性 从天然蛋白质中提取的多肽的形成，只是假设 而且还没有对这些规则进行直接测试。可得性 两个相关的模型蛋白质系统，再加上对分离的 来自这些蛋白质的螺旋多肽，将提供第一个直接测试 关于蛋白质稳定性的分层模型。 细菌中的hpr蛋白是这些研究的理想载体，因为 来自大肠杆菌和枯草芽孢杆菌的蛋白质是 可用于生物物理分析的足够数量。这两个 蛋白质采用相同的三维结构，尽管只有 34%的序列同源性(86个残基中的30个)。因此，HPR系统 提供了在两个阶段测试分层模型的独特机会 不同的蛋白质和比较“上下文依赖”的结构 队形。代表这些蛋白质螺旋部分的多肽 在水中以孤立的多肽形式显示出大量的螺旋结构。 暴露在溶剂中的螺旋稳定相互作用将在 两个相关蛋白及其与分离多肽结果的比较 为了得到蛋白质和蛋白质的能量学的完整特征 多肽稳定性。HPR蛋白的其他片段将用于 阐述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 二级结构元素。这30个角色中有许多是相同的 定义HPR折叠的两个蛋白质之间的残基将是 是通过定向突变研究确定的。长期目标是 确定每个氨基酸残基对稳定性的贡献 和HPR的结构。突变的结构分支，在 完整的蛋白质和多肽片段都将通过核磁共振确定 光谱学与氢交换相结合来关联 局部构象和整体构象突变的结构效应 稳定性。到目前为止，还没有一个单一的系统提供了这样的机会 将分离的多肽的结构形成结果与 完整的蛋白质，并确定相同突变对 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.