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

重复蛋白和共有蛋白：稳定性、协同性、功能、

• 批准号: 10159263
• 负责人: DOUGLAS E. BARRICK
• 金额: $ 34.82万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-01 至 2022-09-19
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10159263
• 关键词: Address; Affinity; Amino Acid Sequence; Anecdotes; Area; Beds; Binding; Binding Proteins; Biochemistry; Biological; Biophysics; Biotechnology; Catalysis; Conflict (Psychology); Consensus; Coupling; DNA; Data Set; Disease; Distant; Electrostatics; Elements; Enzymes; Equilibrium; Evolution; Exhibits; Family; Free Energy; Frustration; Kinetics; Ligand Binding; Linear Models; Maps; Measures; Medicine; Methods; Modeling; Modernization; Mutagenesis; Nature; Outcome; Pathway interactions; Peptides; Pharmaceutical Preparations; Plant Roots; Principal Investigator; Process; Protein Array; Protein Dynamics; Protein Engineering; Proteins; Reagent; Sequence Alignment; Series; Shapes; Solvents; Specificity; Structure; System; Testing; Thermodynamics; Variant; Work; X-Ray Crystallography; base; design; enzyme activity; globular protein; improved; insight; novel; programs; protein folding; protein structure; reconstruction; therapeutic protein

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.

项目概要 主要目标是 1) 确定实现蛋白质稳定性的机制， 特别是，蛋白质的远距离片段的稳定性如何相互影响，从而产生 使用天然和设计的重复蛋白质的组合来实现协同性，以及 2) 理解并利用基于系统发育的共识方法来设计高通量蛋白质 稳定性和高水平的活性。这些目标将通过三个具体目标来实现 目标。 1）应用我们为自然重复开发的最近邻一维伊辛形式主义 蛋白质来量化局部折叠和最近邻耦合能量到一系列从头开始 贝克实验室设计了螺旋重复蛋白。同时，我们将测量折叠动力学， 使用伊辛分析得出的能源格局框架作为框架 解释。与天然重复蛋白质的比较将揭示之间的折叠差异 设计的和天然的蛋白质。 2）应用我们曾经使用过的共识设计方法 将线性重复蛋白稳定为不同折叠、大小和功能的球状蛋白，以及 3) 确定生物活性维持的程度。在目标 2 中，我们确定了 十六个目标，其中六个取得了良好的初步成果。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 将使用序列和结构指标剖析增加稳定性的基础，并比较 采用“祖宗重建”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， 动态变化是否是普遍相关的。 所有这三个目标都将使用不同蛋白质之间的大量比较 建立统计上严格且总体的设计和共识特征图，允许 我们进行概括，确定哪些有效，哪些无效以及原因。这是一个意义重大的 比迄今为止描述的一次性轶事研究有所改进。 实现这些目标将加深我们对自然限制的理解 发生的蛋白质序列和进化，并将解决几个范式，包括“ 最小挫折原则”和“稳定性与活动权衡”，并将确定关键 从头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 提供对蛋白质折叠更深入、更完整的理解，也将提高我们的 为生物技术和医学设计蛋白质的能力。