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%的蛋白质是由模块化的内部重复单元组成的。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、周期性的结构和短距离相互作用的优势极大地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，含有锚蛋白重复序列（33个残基序列基序）的蛋白构成了最具特征和最大的家族之一。该资助的具体目的包括使用基于核磁共振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组成的致心肌激素。肌营养因子之所以被选为模型系统，是因为它折叠良好，可在广泛的条件下溶解。本研究的目的是确定折叠起始位点，并提供折叠过程的残基特异性图像。为了进一步简化分子相互作用的复杂性，我们设计并表征了一系列具有相同重复序列的一致性锚蛋白重复序列。预计通过尿素变性、荧光标记和有限蛋白水解对这些蛋白质的研究将最终使我们能够建立一个关于锚蛋白重复序列折叠的理论模型。最后，我们将探讨基于锚蛋白重复序列的分子识别规则，使用共识锚蛋白重复序列蛋白作为支架来设计特定的结合相互作用。总之，这些联合的方法将产生关于多个重复蛋白折叠的重要信息，以及结构和折叠异常如何强调严重的人类疾病。

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.