CONFORMATIONS OF LIGANDS AT ANTIBODY BINDING SITES

这项研究将介绍当代单克隆抗体提供的工具， 单克隆抗体（Mab）技术引入配体-大分子物理研究 约束力 大量结构上的有利使用 Fab片段形式的同质抗体结合位点 单克隆免疫球蛋白的研究构成了这项工作的基础。 我们计划 一系列旨在阐明，在亚分子 详细地，一些生物学功能半抗原与单克隆抗体的结合， 产生了针对它们及其结构类似物的抗体。 两 选择用于初始研究的半抗原系统是阿片类药物和 趋化性三肽 在初步工作中，我们已经获得了4条线， 针对非重要表位的单克隆抗体 并证明了它们的低解离常数， 吗啡及其对阿片激动剂的不同交叉反应性， 对手。 我们的一位合作者在 趋化性三肽系统，该项目现已被接管到 我们的实验室 物理方法的选择是高频核 磁共振用于溶液研究，中子衍射用于 结晶的半抗原-Fab复合物。 在解决方案中，使用来自 微观酸度常数、化学位移和质子-质子 半抗原和免疫球蛋白共振的Overhauser效应 片段，我们将形成连接免疫球蛋白中残基的邻近图 半抗原上的原子折叠。 借助特定的氘代 半抗原的衍生物，我们将确定它们的构象， 利用差中子衍射的自身和交叉反应结合位点 在晶体复合物上进行。 这项工作将与 轻链和轻链高变区的氨基酸序列分析 免疫球蛋白片段的重链。 标记、衍射和 测序工作将与专家合作进行， investigators. 这项工作将显着不同于以往的工作大分子 结合位点。 我们将研究生物功能配体 其中Mab工具允许我们检查 大分子结合位点的详细描述。 此外，还有良好的 在几个系统中出现的证据，包括趋化性 肽一，抗体结合位点可能是很好的模型， 呈现给半抗原的细胞受体结合位点，例如我们将 在正常的生物功能中学习。

This research will introduce tools offered by contemporary monoclonal antibody (Mab) technology into physical studies of ligand-macromolecular binding. The advantageous use of the large quantities of structurally homogeneous antibody binding sites available in the form of Fab fragments of monoclonal immunoglobulins forms the basis of the work. We plan a series of experiments which are aimed at elucidating, in submolecular detail, the binding of some biologically functional haptens to monoclonal antibodies raised against them and their structural analogs. The two haptenic systems chosen for initial study are the opiates and the chemotactic tripeptides. In preliminary work we have obtained 4 lines of monoclonal antibodies directed against pharmacologically important epitopes on morphine and have demonstrated their low dissociation constants from morphine and their differential cross-reactivities to opiate agonists and antagonists. One of our collaborators has done similar work in the chemotactic tripeptide system and this project has now been taken over into our laboratory. The physical methods of choice are high frequency nuclear magnetic resonance for solution studies and neutron diffraction for crystallized hapten-Fab complexes. In solution, using information from microscopic acidity constants, chemical shifts and proton-proton Nuclear Overhauser Effects for resonances from both haptens and immunoglobulin fragments we will form proximity maps connecting residues in immunoglobulin folds with atoms on the haptens. With the aid of specifically deuterated derivatives of the haptens we will determine their conformations at their own and cross-reacting binding sites using difference neutron diffraction performed on crystalline complexes. This work will be correlated with amino acid sequence analysis of the hypervariable regions of the light and heavy chains of the immunoglobulin fragments. Labeling, diffraction and sequencing work will each be done in collaboration with expert investigators. This work will differ significantly from previous work on macromolecular binding sites. We will be working with biologically functional ligands where the Mab tool allows us to examine the fine structures of the macromolecular binding sites in a detailed way. In addition, there is good evidence which is emerging in several systems, including the chemotactic peptide one, that antibody binding sites may be good models for the cellular receptor binding sites presented to haptens, such as we will be studying during their normal biological functions.