Determination of the basis of ligand binding via engineering and crystallography

通过工程和晶体学确定配体结合的基础

• 批准号: 9134178
• 负责人: BARRY L. STODDARD
• 金额: $ 34.76万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9134178
• 关键词: Address; Affinity; Algorithms; Avidin; Behavior; Binding; Binding Proteins; Binding Sites; Biological Phenomena; Biological Process; Calorimetry; Chemicals; Circular Dichroism; Competitive Binding; Complex; Complex Mixtures; Crystallography; Dissection; Drug Design; Engineering; Ensure; Equilibrium; Foundations; Free Energy; Gene Expression Regulation; Health; Immune; Individual; Lead; Ligand Binding; Ligands; Literature; Measures; Metabolism; Methods; Modeling; Molecular Biology; Mutagenesis; Outcome; Pathway interactions; Pattern; Performance; Play; Process; Protein Engineering; Proteins; Research Personnel; Resolution; Role; Sampling; Sequence Analysis; Shapes; Signal Transduction; Solvents; Specificity; Streptavidin; Structure; System; Thermodynamics; Time; Titrations; Validation; base; design; design and construction; high throughput screening; improved; interest; iterative design; novel; pressure; screening; theories

 DESCRIPTION (provided by applicant): A significant problem in molecular biology is our inability to accurately calculate, model and predict protein-ligand binding affinities, even when provided a high-resolution structure of the actual complex. This problem is made evident across a wide body of experimentation and literature, including (1) the poor performance of algorithms used to calculate binding affinities from structures, (2) disagreement on the physical basis for high affinity ligand binding for exceptionally well-studied proteins such as streptavidin, and (3) difficulties associated with engineering novel ligand-binding proteins. Attempts to understand the basis for tight, specific ligand binding by dissecting naturally evolved ligand binding protein, while informative, have not produced the ability to accurately predict binding affinities from structures, or to directly compute the structure of novel ligand binding proteins. Engineered proteins offer a possible advantage as alternative systems for the examination of ligand binding mechanisms. While the energetic and structural parameters that are used to create them may be inaccurate, those terms are nonetheless precisely defined during the engineering process and can be systematically altered during an iterative design project. In addition, protein engineering allows investigators to create large numbers of designed proteins against many precisely defined ligands, and then to identify the most interesting and informative constructs for detailed structural and physical analyses. The Specific Aims of this project are: (1) To determine common structural and mechanistic features of successfully designed ligand binding proteins, and to compare those results against designs that display unexpected patterns of ligand binding affinities and specificities. (2) To assess whether constraints that are placed on the design of ligand binding proteins behave as intended. In particular, this study will examine whether two fundamental components of ligand- protein designs (shape complementarity, which attempts to enforce specificity, and structural 'pre-ordering' of the binding site, which attempts to reduce entropic penalties) actually impair ligand-binding function. We believe that completion of these aims will provide both an immediate impact (by improving protein design methods) and a longer-term impact (by further elucidating rules that govern the behavior of ligand binding proteins in general).

 描述(申请人提供)：分子生物学中的一个重要问题是，即使提供了实际复合体的高分辨率结构，我们也无法准确地计算、建模和预测蛋白质-配体结合亲和力。这个问题在广泛的实验和文献中都是显而易见的，包括(1)用于从结构计算结合亲和力的算法的糟糕性能，(2)对于特别研究得特别好的蛋白质，如链霉亲和素，在高亲和力配体结合的物理基础上的分歧，以及(3)与工程新的配体结合蛋白相关的困难。试图通过解剖自然进化的配体结合蛋白来了解紧密、特定的配体结合的基础，虽然提供了信息，但并不能从结构中准确地预测结合亲和力，或直接计算新的配体结合蛋白的结构。工程蛋白作为研究配体结合机制的替代系统提供了可能的优势。虽然用于创建它们的能量和结构参数可能不准确，但这些术语在工程过程中是精确定义的，并且可以在迭代设计项目期间进行系统更改。此外，蛋白质工程允许研究人员根据许多精确定义的配体创建大量设计的蛋白质，然后确定最有趣和最有信息量的结构 详细的结构和物理分析。本项目的具体目标是：(1)确定成功设计的配体结合蛋白的共同结构和机制特征，并将这些结果与显示意外的配体结合亲和力和特异性的设计进行比较。(2)评估对配体结合蛋白的设计施加的限制是否如预期的那样起作用。特别是，这项研究将检查配体-蛋白质设计的两个基本组成部分(试图加强特异性的形状互补和试图减少熵惩罚的结合位点的结构预排序)是否实际上损害了配体结合功能。我们相信，这些目标的完成将提供即时影响(通过改进蛋白质设计方法)和长期影响(通过进一步阐明管理一般配体结合蛋白质行为的规则)。