REPEAT-PROTEINS; STABILITY, FOLDING KINETICS & EVOLUTION

描述（由申请人提供）：在生命的所有分支中，有各种蛋白质包含短的串联重复序列。每个重复序列折叠成一个包含螺旋、β链或二级结构单元混合的闭环。然后，相邻的重复序列相互堆叠，形成一个细长的线性域，具有规则的模块化架构。这些重复蛋白通常参与蛋白-蛋白相互作用，通常处于重要生物学过程（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那些难以用非重复蛋白解决的问题。我们将确定重复序列可以相互耦合的距离，并测量这些蛋白质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，我们将通过研究重复蛋白片段的折叠动力学，并确定哪些重复序列在折叠的限速步骤中构成，来研究这种“能量景观”与折叠率的关系，并将测试将折叠率与各种结构和能量特征联系起来的模型。由于其线性、重复的结构，重复蛋白似乎是通过插入和删除快速进化的目标。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。这种重新排列的蛋白质是稳定的，这一发现将支持这样一种观点，即编码重复蛋白的基因重组可以产生功能多样性。我们还将通过对蛋白质序列和结构的计算机分析，寻求对重复蛋白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.