Structure-Function Study of Protein-Protein Recognition

蛋白质-蛋白质识别的结构-功能研究

• 批准号: 6371207
• 负责人: Roy A Mariuzza
• 金额: $ 28.12万
• 依托单位: UNIVERSITY OF MD BIOTECHNOLOGY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-19 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6371207
• 关键词: Escherichia coli; T cell receptor; X ray crystallography; antiantibody; antigen antibody reaction; biochemical evolution; chemical stability; conformation; crystallization; hydropathy; intermolecular interaction; lysozyme; major histocompatibility complex; microcalorimetry; model design /development; natural killer cells; physical model; protein binding; protein purification; protein structure function; site directed mutagenesis; structural biology; superantigens; thermodynamics

DESCRIPTION (provided by applicant): Knowledge of the molecular basis of protein-protein recognition is essential for understanding protein function, since the ability of proteins to form specific complexes with other proteins underlies most cellular processes. Our aim is to progress from purely anatomical descriptions of protein-protein interfaces to an understanding of how structural features contribute to the affinity and specificity of binding reactions. Dr. Mariuzza will carry out detailed structure-function studies of four distinct protein-protein recognition systems: 1) antigen-antibody, 2) natural killer (NK) cell receptor-MHC class I, 3) superantigen (SAG)-MBC class II, and 4) SAGT cell receptor (TCR). For each system, procedures have been developed for the expression of mutant proteins, the crystallization and structure determination of mutant complexes, and the measurement of thermodynamic binding parameters. The following aspects of the association process will be addressed: 1. Energetics of individual interactions across protein-protein interfaces. Double mutant cycles will be constructed to dissect coupling energies between residue pairs in antigen-antibody and NK receptor-MHC class I complexes. Unlike most interfaces described to date (including antigen-antibody), the NK receptorMHC class I interface is highly hydrophilic and dominated by charged interactions. It may represent a qualitatively different strategy that certain proteins have evolved for binding other proteins. 2. Structural basis for specificity in protein-protein recognition. The PI will determine the crystal structures of members of a family of antibodies that bind the immunizing antigen at coincident sites and with similar affinities, but which display striking differences in specificity towards a panel of antigenic variants. 3. Context dependence of the hydrophobic effect in protein-protein interfaces and the nature of energetic "hot spots." Using X-ray crystallography and titration calorimetry, the PI will examine the role that local environment and position in the interface play in determining the hydrophobic contribution of individual residues to binding. The results will improve our understanding of "hot spots" in protein-protein interfaces. 4. Structural determinants of affinity in the evolution of protein-protein interfaces. In order to clarify the structural mechanisms underlying affinity maturation, the PI will determine the crystal structures of antigenantibody, SAG-MHC class II, and SAG-TCR complexes that have acquired up to 10,000-fold increased affinities through directed evolution.

描述（由申请人提供）：蛋白质-蛋白质识别的分子基础知识对于理解蛋白质功能至关重要， 因为蛋白质与其他蛋白质形成特定复合物的能力 是大多数细胞过程的基础。我们的目标是从单纯的进步 蛋白质-蛋白质界面的解剖学描述，以了解 结构特征如何影响结合的亲和力和特异性 反应。 Mariuzza 博士将进行详细的结构功能研究 四种不同的蛋白质-蛋白质识别系统：1) 抗原-抗体，2) 自然杀伤（NK）细胞受体-MHC I类，3）超抗原（SAG）-MBC类 II、和4)SAGT细胞受体(TCR)。对于每个系统，程序已 为突变蛋白的表达、结晶和 突变体复合物的结构测定和测量 热力学结合参数。协会的以下几个方面 流程将被解决： 1. 蛋白质-蛋白质界面上个体相互作用的能量。 将构建双突变体循环来剖析之间的耦合能 抗原-抗体和 NK 受体-MHC I 类复合物中的残基对。不像 迄今为止描述的大多数界面（包括抗原-抗体），NK 受体 MHC I 类界面具有高度亲水性并以带电为主 互动。它可能代表了一种本质上不同的策略，某些 蛋白质已经进化为结合其他蛋白质。 2.蛋白质-蛋白质识别特异性的结构基础。 PI 将 确定结合的抗体家族成员的晶体结构 免疫抗原位于重合位点且具有相似的亲和力，但是 其对一组抗原的特异性表现出显着差异 变体。 3. 蛋白质-蛋白质界面疏水效应的背景依赖性 以及充满活力的“热点”的本质。使用 X 射线晶体学和 滴定量热法，PI 将检查当地环境和 界面中的位置在决定疏水性贡献方面发挥作用 单个残基结合。结果将加深我们的理解 蛋白质-蛋白质界面中的“热点”。 4. 蛋白质-蛋白质进化中亲和力的结构决定因素 接口。为了阐明亲和力背后的结构机制 成熟时，PI将确定抗原抗体的晶体结构， SAG-MHC II 类和已获得高达 10,000 倍的 SAG-TCR 复合物 通过定向进化增加亲和力。