THERMODYNAMIC BASIS OF ANTIBODY-ANTIGEN AFFINITY

抗体-抗原亲和力的热力学基础

• 批准号: 3468120
• 负责人: Richard Willson
• 金额: $ 13.84万
• 依托单位: UNIVERSITY OF HOUSTON
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-09-01 至 1995-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3468120
• 关键词: X ray crystallography; affinity chromatography; antibody formation; antigen antibody reaction; binding proteins; immune complex; lysozyme; molecular dynamics; mutant; protein sequence; site directed mutagenesis; thermodynamics

Recent crystallographic studies have revealed the detailed structure of several antibody/protein antigen complexes. What remains to be understood is the relationship between geometric proximity of potentially=interacting groups, as observed in the complex, and the affinity and selectivity of molecular recognition of the antigen by the antibody. The relationship between structure and molecular recognition is complicated by dynamical motions of residues in the contact region and electrostatic interactions in the complex dielectric environment of the protein/protein interface. It is desireable, therefore, to take a molecular-scale, thermodynamic approach to the structural basis of antibody/antigen affinity. We will use one of the few antibody/protein complexes of known structure, that formed by hen egg lysozyme with the FAb HyHEL-5, for a detailed study of the structural and thermodynamic basis of antibody binding affinity. Directed mutagenesis of the antigen by us, and of the antibody by our collaborator Dr. S. Smith-Gill of the N.I.H., will allow detailed testing of the contributions of specific residues and contacts to the affinity and specificity of binding. These contributions will be characterized in terms of their effects on binding affinity, and also their effects on the enthalpy of binding using titration microcalorimetry. This combined approach will help to reveal the basic thermodynamic driving forces for molecular recognition in this complex. Free energy simulation will be used to predict the relative affinities and enthalpies of binding of the various mutant complexes, and will also provide insight into the role of protein dynamics and conformational changes in the recognition process. Comparison of the simulation results with experiment will also assist in the further development of the theoretical methods. The significant of the proposed work lies in its potential to advance our understanding of the fundamental basis of molecular recognition. The proposed work represents the first integrated study of a single protein/protein complex of known structure applying the methods of directed mutagenesis, calorimetry, and molecular simulation in a synergistic way. For this reason, we believe that the proposed work has the potential to produce new, general insights into the basis of molecular recognition in antigen/antibody complexes.

最近的晶体学研究揭示了其详细结构 几种抗体/蛋白质抗原复合物。 还有什么需要理解的 是潜在=交互的几何接近度之间的关系 在复合物中观察到的基团，以及亲和力和选择性 抗体对抗原的分子识别。 关系 结构和分子识别之间的动力学使结构变得复杂 接触区域中残基的运动和静电相互作用 蛋白质/蛋白质界面的复杂介电环境。 这是 因此，需要采用分子尺度的热力学方法来 抗体/抗原亲和力的结构基础。 我们将使用已知结构的少数抗体/蛋白质复合物之一， 由鸡蛋溶菌酶与 Fab HyHEL-5 形成，用于详细研究 抗体结合亲和力的结构和热力学基础。 我们对抗原进行定向诱变，我们对抗体进行定向诱变 NIH 的合作者 S. Smith-Gill 博士将进行详细测试 特定残基和接触对亲和力的贡献 结合的特异性。 这些贡献的特点是 它们对结合亲和力的影响，以及它们对 使用滴定微量热法测定结合焓。 这结合了 方法将有助于揭示基本的热力学驱动力 该复合物中的分子识别。 自由能模拟将用于预测相对亲和力和 各种突变体复合物的结合焓，也将 深入了解蛋白质动力学和构象的作用 识别过程的变化。 模拟结果比较 实验也将有助于该技术的进一步发展 理论方法。 拟议工作的重要性在于它有可能推进我们的工作 了解分子识别的基本原理。 这 拟议的工作代表了对单一研究的首次综合研究 已知结构的蛋白质/蛋白质复合物应用定向方法 以协同方式进行诱变、量热法和分子模拟。 因此，我们相信拟议的工作有潜力 对分子识别的基础产生新的、普遍的见解 抗原/抗体复合物。