Folding of Alpha-Beta Proteins at High Resolution

描述(申请人提供)：这项建议的目标是阐明两个α-β蛋白的折叠机制，核糖体蛋白L9的N-末端结构域(NTL9)和L9的C-末端结构域(CTL9)。NTL9是一类重要的常见层状片状螺旋结构的较简单的例子之一。CTL9包含一个有趣的混合并行反并行测试页。将解决的关键问题包括变性状态的性质以及变性状态结构对折叠和稳定性的影响。过渡状态有多宽或多窄？折叠的过渡状态的详细性质是什么？突变分析是否提供了过渡状态的准确图景？这项工作提供的见解有望对生物技术、生物物理学和基础生物医学产生重大影响。 静电相互作用在NTL9的变性状态中起着关键作用。对一组突变体的分析将确定它们的来源。调控这些相互作用对折叠的影响将通过动力学研究和突变分析相结合来确定。将对含有天然和非天然氨基酸的变体进行一系列动力学折叠实验，以提供过渡状态下侧链相互作用的细节信息。同位素效应实验将提供有关过渡态骨架结构的关键信息。协作工作将使我们能够将我们的结果与计算研究的预测进行比较。这些实验将为折叠提供一个独特的高分辨率过渡态视图。将对CTL9的折叠进行研究。还没有对这种类型的基序进行折叠研究。掩埋的极性相互作用在CTL9折叠中的作用将被阐明，并通过诱变和pH依赖的研究来检测变性状态的性质。突变分析将被用来绘制折叠的过渡态。其具体目的是1)阐明NTL9变性状态下的静电相互作用2)确定它们在折叠中的作用3)开发NTL9折叠的过渡态的高分辨率视图4)开发CTL9折叠的详细图像。

DESCRIPTION (provided by applicant): The goal of this proposal is to elucidate the folding mechanism of two alpha-beta proteins, the N-terminal domain of the ribosomal protein L9 (NTL9) and the C-terminal domain of L9 (CTL9). NTL9 is one of the simpler examples of an important common class of layered sheet-helix structures. CTL9 contains an interesting mixed parallel anti-parallel beta-sheet. Key questions that will be addressed include the nature of the denatured state and the effects of denatured state structure on folding and stability. How broad or narrow is the transition state? What is the detailed nature of the transition state for folding? Does mutational analysis provide an accurate picture of the transition state? The insights provided by this work are expected to have significant impact on biotechnology, biophysics and basic biomedicine. Electrostatic interactions play a key role in the denatured state of NTL9. Analysis of a set of mutants will determine their origin. The effect of modulating these interactions on folding will be determined by combining kinetic studies with mutational analysis. A set of kinetic folding experiments with variants containing natural and unnatural amino acids will be carried out to provide information on the details of sidechain interactions in the transition state. Isotope effect experiments will provide key information about backbone structure in the transition state. Collaborative work will allow us to compare our results with the predictions of computational studies. These experiments will provide a unique high-resolution view of the transition state for folding. The folding of CTL9 will be investigated. No folding studies of this type of motif have been carried out. The role of buried polar interactions in the folding of CTL9 will be elucidated and the properties of the denatured state examined by mutagenesis and pH dependent studies. Mutational analysis will be used to map out the transition state for folding. The specific aims are 1) To elucidate electrostatic interactions in the denatured state of NTL9 2) To determine their role in folding 3) To develop a high resolution view of the transition state for the folding of NTL9 4) To develop a detailed picture of the folding of CTL9.