MEASUREMENT OF CHANGES IN DENATURED STATE ENERGETICS

描述(摘自申请人的摘要)：蛋白质折叠是一种 在生物化学中具有重要意义的问题。预测的能力 蛋白质的一级序列中的蛋白质结构和功能 对蛋白质类药物的发展具有重要意义。 类似的，了解蛋白质折叠的实际机制，可以 导致对分子疾病的更好的理解，并允许设计 避免折叠陷阱的蛋白质药物。蛋白质稳定其自身 三维结构。然而，更少的人了解 另一半蛋白质折叠平衡，处于变性状态。核磁共振 研究揭示了这种状态的一些结构特性。 然而，人们对结构性变化之间的关系知之甚少 和自由能，在这种松散定义的状态下。这个实验室最近 开发了一种评估突变引起的变性状态自由能的方法 改变。该方法包括测量In的粘结强度 组氨酸-血红素在变性的iso-1-细胞色素c中的连接。在这个提议中， 这项技术将用于： 评估义齿随机线圈行为的偏差 ISO-1-细胞色素 C与组氨酸在序列中的不同位置上 亚铁血红素。 评估第二个站点变异的影响，包括近距离和远距离 由负责组氨酸-亚铁血红素连接的组氨酸变性 ISO-1-细胞色素c. 使用小的血红素多肽来评估局部和长期对 变性状态稳定性。 评价变性态自由能与变性剂的关系 集中精神。这套实验将提供急需的 关于变性蛋白质的能量格局的知识，这将是 在定义蛋白质折叠方面非常重要。

DESCRIPTION (Adapted from applicant's abstract): Protein folding is a problem of great significance in biochemistry. The ability to predict protein structure and function from the primary sequence of a protein would be of great importance tothe development of protein-based pharmaceuticals. Similar, knowledge of the actual mechanism by which proteins fold, could lead to a better understanding of molecular diseases and allow the design of protein pharmaceuticals which avoid folding traps. protein stabilize its three-dimensional structure. However, much less in understood about the other half of the protein folding equilibrium, the denatured state. NMR studies have shed light on some of the structural properties of this state. However, little is known about the relationship between structural changes and free energy in this loosely defined state. This laboratory has recently developed a means of assessing mutation-induced denatured stated free energy changes. The method involves measurement of in the bond strength of histidine-heme ligation in denatured iso-1-cytochrome c. In this proposal, this technique will be used to: evaluate deviations in random coil behavior for denture iso-1-cytochromes c with histidine at different positions in the sequence with respect to the heme. evaluate the consequences of second site variants both near to and far from the histidine responsible for histidine-heme ligation in denatured iso-1-cytochrome c. use small heme-peptides to evaluate local versus long-range effects on denatured state stability. assess the dependence of denatured state free energy on denaturant concentration. This set of experiments will provide much needed knowledge about the energy landscapes of denatured proteins, which will be of great importance in defining the protein folding.