REPEAT-PROTEINS; STABILITY, FOLDING KINETICS & EVOLUTION

描述（由申请人提供）：在生命的所有分支中，存在多种含有短的串联重复序列的蛋白质。每个重复序列折叠成含有螺旋、β链或二级结构单元的混合物的闭环。然后相邻的重复序列彼此堆叠，以产生具有规则的模块化结构的伸长的线性结构域。这些重复蛋白通常参与蛋白质-蛋白质相互作用，通常处于重要生物过程如信号转导的中心，并且通常与疾病状态如癌症和细菌发病机制相关。在这里概述的研究中，将对重复蛋白进行生物物理研究，以更好地了解它们的折叠和稳定性。我们的实验将利用这些蛋白质的线性结构来回答非重复蛋白质难以解决的问题。我们将确定重复可以相互耦合的距离，并测量这些蛋白质中的端到端稳定性分布。我们将使用删除实验来构建具有单重复分辨率的“能量景观”。然后，我们将研究这种“能量景观”如何涉及到折叠速率通过研究重复蛋白质片段的折叠动力学，并通过确定哪些重复结构中的限速步骤折叠，并将测试模型，折叠速率与各种结构和充满活力的功能。由于其线性的重复结构，重复蛋白似乎是通过插入和缺失快速进化的目标。我们将通过研究不同类型的缺失和重复对稳定性的影响来评估这种重排的结果。我们将测试不同类型的重复蛋白的融合是否可以采用稳定的折叠。这种重排蛋白是稳定的这一发现将支持这样的想法，即功能多样性可以通过编码重复蛋白的基因重组产生。我们还将通过对蛋白质序列和结构的计算机分析来寻求对重复蛋白质在蛋白质进化早期出现的假设的支持。

DESCRIPTION (provided by applicant): In all branches of life, there are a variety of proteins that contain short, tandemly repeated sequences. Each repeat-sequence folds up into a closed loop containing helices, beta-strands, or a mix of secondary structure units. Adjacent repeats then pack against each other to create an elongated, linear domain with a regular, modular architecture. These repeat-proteins usually participate in protein-protein interactions, often at the center of important biological processes such as signal transduction, and are often associated with disease states such as cancer and bacterial pathogenesis. In the research outlined here, biophysical studies will be performed on repeat-proteins to better understand their folding and their stability. Our experiments will take advantage of the linear architecture of these proteins to answer questions that are difficult to address with non-repeat proteins. We will determine the distances over which repeats can couple with one another, and measure the end-to-end stability distribution in these proteins. We will use deletion experiments to build an "energy landscape" with single-repeat resolution. We will then examine how this "energy landscape" relates to folding rates by studying the folding kinetics of repeat-protein fragments and by determining which repeats are structured in the rate-limiting steps in folding, and will test models that relate folding rates to various structural and energetic features. Owing to their linear, repeated architecture, repeat-proteins seem likely targets for rapid evolution through insertion and deletion. We will evaluate the outcome of such rearrangements by studying the effects on stability of different types of deletions and duplications. We will test whether the fusion of different types of repeat-proteins can adopt a stable fold. The finding that such rearranged proteins are stable would support the idea that functional diversity can be produced by recombination of genes encoding repeat-proteins. We will also seek support for the hypothesis that repeat-proteins appeared early in protein evolution by computer analysis of protein sequences and structures.