MEASUREMENT OF CHANGES IN DENATURED STATE ENERGETICS

描述（改编自申请人的摘要）：蛋白质折叠是一种 生物化学中的一个重要问题。 风暴预测 从蛋白质的一级序列来分析蛋白质的结构和功能， 对蛋白质药物的开发具有重要意义。 类似地，对蛋白质折叠的实际机制的了解， 从而更好地理解分子疾病， 避免折叠陷阱的蛋白质药物。 蛋白质稳定其 三维结构。 然而，很少有人了解 蛋白质折叠平衡的另一半，即变性状态。 NMR 研究揭示了这种状态的一些结构特性。 然而，人们对结构变化之间的关系知之甚少。 和自由能之间的关系 该实验室最近 开发了一种评估突变诱导的变性状态自由能的方法 变化 该方法包括测量粘合强度， 变性异-1-细胞色素c中组氨酸-血红素连接。在这一提议中， 这项技术将用于： 评价义齿随机线圈行为的偏差 异-1-细胞色素 c在序列中的不同位置具有组氨酸， 血红素 评估第二个站点变体的后果，包括近距离和远距离 从负责变性的组氨酸-血红素连接的组氨酸 异-1-细胞色素c。 使用小血红素肽来评估局部与远程影响， 变性状态稳定性 评估变性状态自由能对变性剂的依赖性 浓度. 这组实验将提供急需的 关于变性蛋白质的能量景观的知识，这将是 对蛋白质折叠的定义非常重要。

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.