THERMODYNAMIC BASIS OF ANTIBODY-ANTIGEN AFFINITY

抗体-抗原亲和力的热力学基础

• 批准号: 2182486
• 负责人: Richard Willson
• 金额: $ 8.39万
• 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/2182486
• 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 几种抗体/蛋白抗原复合物。 还有什么需要了解的 是潜在相互作用的几何接近度之间的关系 基团，如在复合物中观察到的，以及 抗体对抗原的分子识别。 的关系 结构和分子识别之间的关系是复杂的动力学 残基在接触区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。 是 因此，希望采取分子尺度的热力学方法， 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， 用鸡蛋溶菌酶与FAb HyHEL-5形成的蛋白质，进行详细研究 抗体结合亲和力的结构和热力学基础。 我们对抗原进行了定向诱变，我们对抗体进行了定向诱变。 合作者S.国家卫生研究所的史密斯-吉尔将允许详细的测试 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， 使用滴定微量热法测定结合焓。 该组合 方法将有助于揭示基本的热力学驱动力， 分子识别 自由能模拟将用于预测相对亲和力， 各种突变体复合物的结合能力，也将 提供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 与实验也将有助于进一步发展的 理论方法 拟议工作的重要性在于它有可能促进我们的 了解分子识别的基本原理。 的 拟议的工作是第一次综合研究一个单一的 已知结构的蛋白质/蛋白质复合物， 诱变，量热法，和分子模拟的协同方式。 因此，我们认为，拟议的工作有可能 产生新的，一般的见解，分子识别的基础， 抗原/抗体复合物。