Folding and Assembly of Ankyrin Repeat Proteins

描述（由申请人提供）：本研究的长期目标是了解多个重复蛋白的折叠和结构组装。基因组测序分析的最新进展表明，多达30%的蛋白质由模块化的内部重复单元组成。这些蛋白质通常具有重要的调节功能，使用重复单元作为识别模块与广泛的蛋白质或非蛋白质靶标相互作用。内部重复蛋白质也为我们提供了一个独特的机会来研究蛋白质折叠的基本原理，因为规则的，周期性的结构和短程相互作用的优势大大简化了分子内耦合网络的拓扑结构。在所有的内部重复序列蛋白中，那些含有锚蛋白重复序列（一个33个残基的序列基序）的蛋白形成了最具特征和最大的家族之一。这项资助的具体目标包括使用基于NMR的尿素变性和氢交换研究来研究肌营养素的折叠机制，肌营养素是一种由四个锚蛋白重复序列组成的心肌激素。选择肌营养蛋白作为模型系统，因为它在广泛的条件下折叠良好且可溶。本研究的目的是确定折叠起始位点，并提供一个残基特异性的折叠过程的图片。为了进一步简化分子间相互作用的复杂性，设计并表征了一系列具有相同重复序列的共有锚蛋白重复序列蛋白。预计这些蛋白质的尿素变性，荧光标记，和有限的蛋白水解的研究将最终使我们能够建立一个理论模型的折叠锚蛋白重复。最后，以锚蛋白重复序列为骨架设计特异性结合作用，探索基于锚蛋白重复序列的分子识别规律。总之，这些结合的方法将产生关于多个重复蛋白质的折叠以及结构和折叠异常如何强调严重的人类疾病的重要信息。

DESCRIPTION (provided by applicant): The long-term goal of this research is to understand the folding and structural assembly of multiple repeat proteins. Recent progress in genomic sequencing analysis indicates that as much as 30% of all proteins are composed of modular, internal repeat units. These proteins often hold important regulatory functions using the repeating units as recognition modules to interact with a wide range of protein or non-protein targets. The internal repeat proteins also provide us with a unique opportunity to study the fundamental principles of protein folding because the regular, periodic structure and the dominance of short-range interactions greatly simplify the topology of the intramolecular coupling network. Among all internal repeat proteins, those containing the ankyrin repeat, a 33-residue sequence motif, form one of the best-characterized and largest families. Specific aims of this grant include the use of NMR-based urea denaturation and hydrogen exchange studies to investigate the folding mechanism of myotrophin, a cardiomyogenic hormone consisting of four ankyrin repeats. Myotrophin was chosen as a model system because it is well-folded and soluble under a broad range of conditions. The goal of this study is to identify the folding initiation sites and to provide a residue-specific picture of the folding process. To further simplify the complexity of molecular interactions, a series of consensus ankyrin repeat proteins with identical repeats have been designed and characterized. It is anticipated that studies of these proteins by urea denaturation, fluorescence labeling, and limited proteolysis will eventually allow us to build a theoretical model on the folding of ankyrin repeats. Finally, the rules of ankyrin repeat based molecular recognition will be probed using the consensus ankyrin repeat proteins as a scaffold to design specific binding interactions. Taken together, these combined approaches will generate important information regarding the folding of multiple repeat proteins and how the structural and folding abnormalities may underline serious human diseases.