Folding of Alpha-Beta Proteins at High Resolution

描述（由申请人提供）：本提案的目的是阐明两种α-β蛋白的折叠机制，即核糖体蛋白L9的N-末端结构域（NTL 9）和L9的C-末端结构域（CTL 9）。NTL 9是一个简单的例子，一个重要的常见类别的分层片螺旋结构。CTL 9含有有趣的混合平行反平行β折叠。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的影响。过渡状态有多宽或窄？折叠过渡态的具体性质是什么？突变分析是否提供了一个准确的过渡状态的图片？这项工作提供的见解预计将对生物技术，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NTL 9的变性状态中起关键作用。对一组突变体的分析将确定它们的起源。调节这些相互作用对折叠的影响将通过将动力学研究与突变分析相结合来确定。将进行一组含有天然和非天然氨基酸的变体的动力学折叠实验，以提供关于过渡态侧链相互作用的细节的信息。同位素效应实验将提供有关过渡态骨架结构的关键信息。合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。这些实验将为折叠过渡态提供一个独特的高分辨率视图。将研究CTL 9的折叠。没有折叠的研究，这种类型的主题已进行。埋藏的极性相互作用在折叠的CTL 9的作用将被阐明，并通过诱变和pH依赖性研究检查变性状态的属性。突变分析将用于绘制折叠的过渡状态。具体目的是：1）阐明NTL 9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NTL 9折叠过渡态的高分辨率视图4）开发CTL 9折叠的详细图片。

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.