Folding of Alpha-Beta Proteins at High Resolution

这一建议的目的是阐明两个O_-(3)蛋白的折叠机制，N-。 核糖体蛋白L9的末端结构域(NTL9)和L9的C末端结构域(CTL9)。NTL9是 这是一类重要的常见层状螺旋结构的较简单的例子之一。第9号货柜码头 包含一个有趣的混合并行反并行I_表。将解决的关键问题 包括变性状态的性质和变性状态结构对折叠和 稳定性。过渡状态有多宽或多窄？过渡的详细性质是什么？ 折叠的状态？突变分析是否提供了过渡状态的准确图景？这个 这项工作提供的见解预计将对生物技术、生物物理学产生重大影响 和基础生物医学。 静电相互作用在NTL9的变性状态中起着关键作用。分析了一组 变种人将决定它们的起源。调节这些相互作用对折叠的影响将是 由动力学研究和突变分析相结合确定的。一套动态折叠实验 含有天然和非天然氨基酸的变种将进行提供信息 关于过渡态中侧链相互作用的细节。同位素效应实验将提供 有关处于过渡状态的主干结构的关键信息。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。这些实验将提供一种 用于折叠的过渡状态的唯一高分辨率视图。九号货柜码头的折叠将是 调查过了。还没有对这种类型的基序进行折叠研究。埋藏的极地的作用 CTL9在折叠过程中的相互作用以及变性状态的性质将被阐明 通过诱变和pH依赖研究进行检测。突变分析将被用来绘制 折叠的过渡状态。具体的目的是1)阐明静电相互作用在 NTL9的变性状态2)以确定它们在折叠中的作用3)以开发高分辨率视图 NTL9折叠的过渡态4)，以开发CTL9折叠的详细图像。

The goal of this proposal is to elucidate the folding mechanism of two o_-(3proteins, 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 I_-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 aminoacids 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.