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; 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，蛋白质遥远段的稳定性如何相互影响，产生 合作，使用天然和设计的重复蛋白的组合，以及2） 了解和利用基于系统的共识方法来设计高的蛋白质 稳定性和高水平的活动。这些目标将通过三个特定的 目标。 1）应用我们为自然重复开发的最近的邻居1D伊辛形式主义 蛋白质以量化局部折叠和最近的邻居耦合能与一系列从头开始 从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， 使用由Ising分析导致的能源景观框架作为框架 解释。与自然重复蛋白的比较将揭示折叠的差异 设计和天然蛋白质。 2）应用我们过去的共识设计方法 将线性重复蛋白稳定到不同折叠，大小和功能的球状蛋白上，3） 确定维持生物学活性的程度。在AIM 2中，我们已经确定 16个目标，并具有六个目标的强烈初步结果。我们将通过 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来剖析稳定性提高的基础，并比较 采用“祖先重建”方法。在AIM 3中，我们将衡量具有约束力的亲和力， 特异性和酶活性，将集中于高稳定性是否会降低活动， 并且动态是否改变是一般相关性。 这三个目标将使用不同蛋白质之间的大量比较 构建设计和共识特征的统计严格和一般图片，允许 我们概括，确定什么有效，什么无效以及为什么。这是重要的 迄今为止已经描述的一次性轶事研究的改进。 实现这些目标将提高我们对自然限制的理解 发生蛋白质序列和进化，将解决包括“ 最小的挫败感原则和“稳定性活动权衡”，将确定关键 从头设计和天然蛋白质序列之间的差异。这些研究会 提供对蛋白质折叠的更深入，更完整的了解，也将改善我们的 能够设计生物技术和医学的蛋白质。