Repeat and Consensus Proteins: Stability, Cooperativity, Function, & Design

项目摘要 广泛的目标是1）确定实现蛋白质稳定性的机制， 特别是蛋白质远距离片段的稳定性如何相互影响， 协同性，使用天然和设计的重复蛋白质的组合，和2） 理解并利用基于遗传学的共识方法来设计蛋白质， 稳定性和高活性。这些目标将通过以下三个具体措施实现： 目标。1)应用我们为自然重复开发的最近邻1D伊辛形式主义 蛋白质，以量化局部折叠和最近邻耦合能量，以一系列从头 贝克实验室设计的螺旋重复蛋白。同时，我们将测量折叠动力学， 利用伊辛分析得出的能源格局框架， 解释。与天然重复序列蛋白的比较将揭示 设计和天然蛋白质。2)应用我们已经使用的共识设计方法， 将线性重复蛋白稳定为不同折叠、大小和功能的球状蛋白，和3） 确定维持生物活性的程度。在目标2中，我们确定了 16个目标，并有六个强有力的初步结果。我们将通过以下方式确定结构 NMR和X射线晶体学，以及使用溶液热力学和动力学的稳定性。我们 将使用序列和结构度量来剖析增加稳定性的基础，并比较 用“祖先重建”的方法。在目标3中，我们将测量结合亲和力， 特异性和酶活性，并将重点关注高稳定性是否会降低活性， 动力学是否改变是一个普遍的相关性。 所有这三个目标都将使用不同蛋白质之间的大量比较 建立一个统计上严格和一般的图片设计和共识的特点，允许 我们去归纳，确定什么有效，什么无效，以及为什么。这是一个重大 与迄今为止描述的一次性轶事研究相比，这是一种改进。 实现这些目标将促进我们对自然环境的限制的理解。 发生的蛋白质序列和进化，并将解决几个范例，包括“ 最小挫折原则”和“稳定-活动权衡”，并将确定关键 新设计的蛋白质序列和天然蛋白质序列之间的差异。这些研究将 提供了更深入和更完整的了解蛋白质折叠，也将提高我们的 为生物技术和医学设计蛋白质的能力。

Project Summary The broad objectives are 1) to determine the mechanisms by which protein stability is achieved, and in particular, how stabilities of distant segments of proteins influence each other, giving rise to cooperativity, using a combination of natural and designed repeat proteins, and 2) to understand and leverage phylogenic-based consensus approaches to design proteins for high stability and high levels of activity. These objectives will be achieved through three specific aims. 1) Apply our nearest-neighbor 1D Ising formalism that we developed for natural repeat proteins to quantify local folding and nearest neighbor coupling energies to a series of de novo designed helical repeat proteins from the Baker lab. In parallel, we will measure folding kinetics, using the energy landscape framework that results from the Ising analysis as a framework for interpretation. Comparison to natural repeat proteins will reveal differences in folding between designed and natural proteins. 2) Apply consensus design methods that we have used to stabilize linear repeat proteins to globular proteins of different folds, sizes, and functions, and 3) determine the extent to which biological activity is maintained. In Aim 2, we have identified sixteen targets, and have strong preliminary results for six. We will determine structures by NMR and x-ray crystallography, and stabilities using solution thermodynamics and kinetics. We will dissect the basis of increased stability using sequence and structure metrics, and compare with the "ancestral reconstruction" approach. In Aim 3, we will measure binding affinities, specificities, and enzyme activities, and will focus on whether high stabilities decrease activity, and whether dynamics changes is a general correlate. All three of these aims will use large numbers of comparisons among different proteins to build a statistically rigorous and general picture of design and consensus features, allowing us to generalize, determining what works, what does not, and why. This is a significant improvement over the one-off anecdotal studies that have been described to date. Achieving these objectives will advance our understanding of the constraints on naturally occurring protein sequences and evolution, and will address several paradigms including the " principle of minimal frustration" and the "stability-activity tradeoff", and will identify key differences between de novo-designed and natural protein sequences. These studies will provide a deeper and more complete understanding of protein folding, and will also improve our ability to design proteins for biotechnology and medicine.