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

基本信息

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
项目类别：
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

项目摘要

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（由申请人提供）：分子生物学中的一个重要问题是我们无法准确计算、建模和预测蛋白质-配体结合亲和力，即使提供了实际复合物的高分辨率结构，这个问题在广泛的领域中都很明显。大量实验和文献，包括（1）用于计算结构结合亲和力的算法性能不佳，（2）对于经过充分研究的蛋白质（例如链霉亲和素）的高亲和力配体结合的物理基础存在分歧， (3) 与工程新型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、特异性配体结合的基础，虽然信息丰富，但尚未产生准确预测结构结合亲和力的能力，或直接计算新型配体结合蛋白的结构作为检查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，但这些术语仍然是精确定义的。在工程过程中，可以系统地此外，在迭代设计项目中，蛋白质工程允许研究人员针对许多精确定义的配体创建大量设计的蛋白质，然后识别最有趣和信息丰富的结构。该项目的具体目标是：(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出意想不到的配体结合亲和力和特异性的设计进行比较2。）为了评估配体结合蛋白设计的限制是否符合预期，本研究将检查配体蛋白设计的两个基本组成部分（形状互补性，试图增强特异性，以及结构“预-”。的排序”结合位点（试图减少熵惩罚）实际上会损害配体结合功能，我们相信，这些目标的完成将提供直接影响（通过改进蛋白质设计方法）和长期影响（通过进一步阐明控制规则）。配体结合蛋白的一般行为）。