Conformational Properties of Protein Denatured States

描述(申请人提供)：蛋白质变性状态仍然知之甚少，然而，作为蛋白质折叠的起点，了解作用于变性状态的构象限制是解决蛋白质如何有效折叠问题的核心。错误折叠的疾病，如阿尔茨海默氏症和帕金森氏病是主要的健康问题，这些疾病的病原体被认为是蛋白质的非天然和变性状态。因此，对变性蛋白质的基础研究对于新的认识这些疾病状态的发生是至关重要的。该实验室开发了一种新的策略来探索未折叠蛋白质的构象和热力学性质。通过组氨酸-血红素环平衡来评估形成不同大小环的倾向。在酵母iso-1-细胞色素c中已经产生了单表面组氨酸变体，允许在变性条件下测量9到83个氨基酸的环的环平衡。当蛋白质折叠时，在结构增长的最早阶段需要形成闭合环。这个系统已经对蛋白质变性状态与随机卷曲行为的偏差产生了重要的见解。该系统将用于研究未折叠蛋白质的几个关键性质。为了了解残基结构如何影响变性蛋白质中的接触概率，我们将用二硫键交联剂稳定残基结构，将已知的稳定的β发夹插入到iso-1-细胞色素c中，并将我们的方法应用于细胞色素c‘，已知它具有比iso-1-细胞色素c更紧密的变性状态(特定目标1)。序列组成对变性状态构象性质的影响将通过在iso-1-细胞色素c(特定目标2)的无序N-末端插入均聚氨基酸序列来探索，重点是柔性氨基酸甘氨酸和刚性氨基酸脯氨酸的性质。计划进行环形成和断裂的动力学研究，以探索环的大小、变性状态致密性和残余结构如何影响变性状态触点形成的速度和导致触点持续存在的因素(具体目标3)。核磁共振和FRET方法将被用来关联变性状态热力学和变性状态结构性质(具体目标4)。我们的多管齐下的方法探索蛋白质变性状态的关键参数，预计这些参数将成为蛋白质折叠早期事件的主要调节器。

DESCRIPTION (provided by applicant): 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 structure impact 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, expected to be principal modulators of early events in protein folding.