MEASUREMENT OF CHANGES IN DENATURED STATE ENERGETICS

描述（改编自申请人摘要）：蛋白质折叠的问题 是非常重要的。蛋白质折叠的两个常见假设 有效地是构象偏好-存在于未折叠状态， 折叠是通过中间体发生的。该提案侧重于更好地 通过仔细的热力学来理解蛋白质的这些非天然状态 问题研究具体来说，调查人员已经开发了一种方法， 允许突变诱导的变性状态的能量学变化， 通过测量细胞色素c的热力学， 在变性条件下形成组氨酸-血红素环。他们还 开发了一种细胞色素c的Lys 73-His变体， 细胞色素c的未折叠状态具有类似于最低能量的性质 H/D交换实验检测到细胞色素c的部分未折叠状态。 因此，它可能是细胞色素晚期折叠中间体的模型 C.这些系统将解决以下问题： 一组血红素-肽模型和组氨酸环的第二位点变体 将产生形成细胞色素C。局部效应与远距离效应 相互作用和构象限制对变性态能量学的影响 会用这些材料来解决。变性态的依赖性 将评估变性剂浓度的能量学，以了解如何变性 当溶液条件有利于折叠时，状态热力学改变。 Trp 59-血红素变性态致密性的实验研究 荧光能量转移将与检测 平衡折叠中间体，以评估中间体与 突变诱导的变性m值变化中的变性状态。 子结构内部与子结构之间的相互作用在稳定 将评估Lys 73-His细胞色素c产生的部分未折叠状态 具有由热力学确定的Δ Cp和其形成的m值 测量值作为pH、温度和变性剂浓度的函数。

DESCRIPTION(adapted from applicant's abstract): The problem of protein folding is of great fundamental importance. Two common postulates for how proteins fold efficiently are that conformational preferences-exist in the unfolded state and that folding occurs through intermediates. This proposal focuses on better understanding these non-native states of proteins through careful thermodynamic studies. Specifically, the investigators have developed a methodology that allows mutation-induced changes in the energetics of the denatured state of cytochrome c to be quantified precisely by measuring the thermodynamics of histidine-heme loop formation under denaturing conditions. They have also developed a Lys73-His variant of cytochrome c which stabilizes a partially unfolded state of cytochrome c having properties similar to the lowest energy partially unfolded state of cytochrome c detected by H/D exchange experiments. It is, thus, potentially a model for a late folding intermediate of cytochrome c. The following issues will be addressed with these systems: A set of heme-peptide models and second-site variants of the histidine loop forming cytochromes c will be produced. The effects of local versus long-range interactions and of conformational restriction on denatured state energetics will be addressed with these materials. The dependence of denatured state energetics on denaturant concentration will be assessed to learn how denatured state thermodynamics change when solution conditions favor folding. Experiments on the compactness of the denatured state using Trp59-heme fluorescence energy transfer will be combined with methods which detect equilibrium folding intermediates to assess the role of intermediates versus denatured states in mutationally-induced changes in denaturation m-values. The role of intra- versus inter-substructure interactions in stabilizing the partially unfolded state produced by Lys73-His cytochrome c will be assessed with delta Cp and the m-value for its formation determined by thermodynamic measurements as a function of pH, temperature, and denaturant concentration.