Conformational Properties of Protein Denatured States

蛋白质的变性状态仍然知之甚少，然而，作为蛋白质折叠的起点， 对作用于变性状态的构象约束的理解是解决这些问题的核心。 蛋白质如何有效折叠的问题。错误折叠疾病，如阿尔茨海默氏症和帕金森氏症 疾病是主要的健康问题，其中的病原体被认为是非本地的， 蛋白质的变性状态因此，对变性蛋白质的基础研究对于新的见解至关重要 这些疾病的起源。该实验室开发了一种新的策略来探测 未折叠蛋白质的构象和热力学性质。形成环的倾向 通过组氨酸-血红素环平衡评估不同的大小。单表面组氨酸变体具有 在酵母iso-1-细胞色素c中产生，允许9至83个氨基酸的环的环平衡， 在变性条件下测量。在最早的阶段， 当蛋白质折叠时结构增加。这个系统已经对偏差产生了重要的见解 蛋白质变性状态的随机卷曲行为。未折叠蛋白质的几个关键特性将是 探索这个系统。为了了解残余结构如何影响变性 蛋白质，我们将用二硫键交联稳定残余结构，插入已知稳定的β发夹， iso-1-细胞色素c，并将我们的方法应用于细胞色素c '，已知细胞色素c'具有更多 比异-1-细胞色素c更紧密的变性状态（具体目标1）。层序组成对 通过插入均聚氨基酸序列， 异-1-细胞色素c的无序N-末端区域（具体目标2），重点是性质 由柔性氨基酸甘氨酸和刚性氨基酸脯氨酸组成。成环动力学研究 断裂计划探测环的大小，变性状态的紧凑性和残余结构的影响 变性状态接触形成的速率和导致接触持续的因素（特定目标 3）。NMR和FRET方法将被用来关联变性状态热力学和变性状态 结构特性（具体目标4）。我们的多管齐下的方法探测蛋白质变性的关键参数 国家预期是蛋白质折叠的早期事件的主要调制器。

Protein denatured states remain poorly understood and yet, as the starting point for protein folding, an understanding of the conformational constraints acting upon the denatured state is central to solving the problem of how a protein folds efficiently. Misfolding diseases,such as Alzheimer's and Parkinson's diseases are major health problems, where the causative agents are believed to be non-native and denatured states of proteins. Thus, fundamental research on denatured proteins is essential to new insight into the genesis of these disease states. This laboratory has developed a novel strategy to probe the conformational and thermodynamic properties of unfolded proteins. The propensity for forming loops of different sizes is assessed through histidine-heme loop equilibria. Single surface histidine variants have been produced in yeast iso-1-cytochrome c, allowing loop equilibria for loops of 9 to 83 amino acids to be measured under denaturing conditions. Formation of closed loops are required in the earliest stages of structure accretion when a protein folds. This system has already yielded important insights into the deviation of protein denatured states from random coil behavior. Several key properties of unfolded proteins will be probed with this system. To understand how residual structure affects contact probability in a denatured protein, we will stabilize residual structure with disulfide crosslinks, insert a known stable beta hairpin into iso-1-cytochrome c, and apply our methodology to cytochrome c', which is know to have a much more compact denatured state than iso-1-cytochrome c (specific aim 1). The effect of sequence composition on denatured state conformational properties will be probed by inserting homopolymeric arnino acid sequences into the disordered N-terminal region of iso-1-cytochrome c (specific aim 2) with emphasis on the properties of the flexible amino acid glycine and the rigid amino acid proline. Kinetics studies on loop formation and breakage are planned to probe how loop size, denatured state compactness and residual structureimpact the rate at which denatured state contacts form and the factors which cause contacts to persist (specific aim 3). NMR and FRET methods will be used to correlate denatured state thermodynamic with denatured state structural properties (specific aim 4). Our multipronged approach probes key parameters of protein denatured states expectedto be principal modulators of early events in protein folding.