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Investigating the Structural Basis of Antibody Steroselectivity (AREA)

研究抗体立体选择性 (AREA) 的结构基础

基本信息

批准号：
7012453
负责人：
HEIKE HOFSTETTER
金额：
$ 21.75万
依托单位：
NORTHERN ILLINOIS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-03-01 至 2010-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7012453
关键词：
X ray crystallography affinity chromatography alcohols aminoacid antibody binding sites chemical group conformation gel filtration chromatography ion exchange chromatography ligands protein structure function site directed mutagenesis stereochemistry stereoisomer transfection /expression vector

项目摘要

DESCRIPTION (provided by applicant): Physiological processes invariably depend on specific interactions between biological binding partners. Since receptors, transport proteins, enzymes, and antibodies are able to differentiate between the stereoisomers of chiral compounds, one stereoisomer is generally preferred in, e.g., metabolic pathways and cellular communication. Thus, living organisms discriminate between the enantiomers of endogenous and exogenous compounds, such as drugs, at virtually all levels of interaction, and respond differently to them. While one enantiomer of a drug may exhibit a desired activity, the other may cause severe pharmacologic and toxicologic side effects, or act as an antagonist. Despite the fact that the stereoselectivity of protein-ligand interactions was first recognized more than a century ago, the molecular basis of such chiral discrimination is not yet understood. Our long-term objective is to elucidate the structural elements that govern chiral discrimination at the molecular level by studying the stereoselective interaction between antibodies and low-molecular weight chiral compounds as a model system. The antigen binding fragments of several antibodies that stereoselectively recognize the D- or L-enantiomers of amino acids or hydroxy acids will be crystallized for structural analysis by X-ray crystallography. While the comparison of the structures of, e.g., anti-D-amino acid antibodies and anti-L-amino acid antibodies in complex with D- and L-amino acids, respectively, will reveal, which residues in the antibody binding site mediate stereoselective interaction with the ligand, the assessment of individual anti-amino acid antibodies with and without ligand will allow us to evaluate the importance of conformational changes for stereoselective binding. Analogous studies with the anti-hydroxy acid antibodies will provide additional information about the effect of substituting functional groups about the stereogenic center. Utilizing the class-specificity of the antibodies under study, structures of selected antibodies in complex with different ligands will disclose the contribution of particular residues to stereoselectivity and overall binding activity. The production of single chain variable fragments and their structure-guided manipulation by site-directed mutagenesis will be used to test the validity of our findings and to create antibodies with tailor-made binding properties. Success in this project will provide new, unprecedented insights into the molecular basis of biorecognition and impact upon both, basic and applied science, and thus human health. A better comprehension of the molecular basis of protein stereoselectivity will, for example, be valuable for the rational design of more efficient drugs and the development of tailor-made chiral host molecules for the analysis, separation, and synthesis of stereoisomeric compounds.

描述（由申请人提供）：生理过程总是依赖于生物结合伙伴之间的特定相互作用。由于受体、转运蛋白、酶和抗体能够区分手性化合物的立体异构体，因此在代谢途径和细胞通讯中，通常优选一种立体异构体。因此，生物在几乎所有的相互作用水平上都能区分内源性和外源性化合物（如药物）的对映体，并对它们作出不同的反应。当药物的一种对映体可能表现出期望的活性时，另一种对映体可能引起严重的药理学和毒理学副作用，或作为拮抗剂。尽管蛋白质-配体相互作用的立体选择性在一个多世纪前就被首次认识到，但这种手性区分的分子基础尚未被理解。我们的长期目标是通过研究抗体和低分子量手性化合物之间的立体选择相互作用作为模型系统，在分子水平上阐明控制手性区分的结构要素。几种立体选择性识别氨基酸或羟基酸的D-或l -对映体的抗体的抗原结合片段将通过x射线晶体学结晶进行结构分析。虽然比较抗D-氨基酸抗体和抗l -氨基酸抗体的结构，例如分别与D-和l -氨基酸复合物，将揭示抗体结合位点的哪些残基介导与配体的立体选择性相互作用，但评估单个抗氨基酸抗体与配体和不含配体的构象变化将使我们能够评估立体选择性结合的重要性。与抗羟基酸抗体类似的研究将提供关于取代立体中心官能团影响的额外信息。利用所研究抗体的类特异性，选择抗体与不同配体的复合物的结构将揭示特定残基对立体选择性和总体结合活性的贡献。单链可变片段的生产及其通过位点定向诱变的结构引导操作将用于测试我们发现的有效性，并创建具有定制结合特性的抗体。这个项目的成功将为生物识别的分子基础提供新的、前所未有的见解，并对基础科学和应用科学以及人类健康产生影响。例如，更好地理解蛋白质立体选择性的分子基础将有助于合理设计更有效的药物，以及开发用于分析、分离和合成立体异构体化合物的量身定制的手性宿主分子。