Folding of Alpha-Beta Proteins at High Resolution

描述（由申请人提供）：本提案的目标是阐明两种α - β蛋白的折叠机制，核糖体蛋白L9的n端结构域（NTL9）和L9的c端结构域（CTL9）。NTL9是一种常见的层状片状螺旋结构的简单例子。CTL9包含一个有趣的混合平行反平行β -片。将解决的关键问题包括变性态的性质以及变性态结构对折叠和稳定性的影响。过渡状态有多宽或多窄？折叠过渡态的具体性质是什么？突变分析是否提供了过渡状态的准确图像？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。

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.