Structure of Antibodies Binding Similar Sites on Cytochromes

将以细胞色素c （cyt）为模型抗原，研究指导抗体识别蛋白质抗原的理化参数。特别感兴趣的是对不同细胞反应的抗体谱差异的基础。单克隆抗体（mab）将被诱导针对几种细胞，这些细胞在抗原区域中只有一个氨基酸的差异。识别的抗原表位将通过抗体结合试验确定，抗体结合试验采用许多由位点定向诱变制备的cyt变体或从自然来源获得。单抗的重链和轻链的氨基酸序列将由cDNA的核苷酸序列推断出来。结合相似表位的单克隆抗体将在初级结构方面进行比较，并且将确定由于表位中氨基酸变化而产生的任何差异。显示这种差异的单克隆抗体的三维结构将使用计算机图形学建模，以及它们与cyt的相互作用，以深入了解相互作用的化学性质。其中一些模型将在相关研究中通过x射线晶体学进行测试。初步数据表明，抗体基因的使用相对有限，不同的V区基因用于响应cyt的两个主要抗原区域。表位形貌对互补区长度的影响也很明显。这些观察结果将进一步研究，以促进我们对天然蛋白抗原的抗体识别的理解。总的来说，这些结果可以作为蛋白质-蛋白质相互作用的模型。

The physicochemical parameters which guide antibody recognition of a protein antigen will be studied employing cytochrome c (cyt) as a model antigen. Of particular interest is the basis for differences in the antibody repertoires responding to variant cyts. Monoclonal antibodies (mAbs) will be elicited against several cyts that differ by as little as one amino acid in an antigenic region. The epitopes recognized will be determined from antibody binding assays employing a number of cyt variants prepared by site-directed mutagenesis or obtained from natural sources. The amino acid sequences of the heavy and light chains of the mAbs will be deduced from the nucleotide sequences of cDNA. mAbs binding similar epitopes will be compared in terms of primary structure and any differences that occur as a result of an amino acid change in an epitope will be determined. The three-dimensional structures of mAbs showing such differences will be modeled using computer graphics as will their interaction with cyt to obtain insight into the chemistry of the interaction. Some of these models will be tested by X-ray crystallography in a related study. Preliminary data indicate relatively restricted antibody gene usage with distinct V region genes used in response to the two major antigenic regions of cyt. An effect of epitope topography on complementary determining region length is also apparent. These observations will be studied further to advance our understanding of antibody recognition of native protein antigens. The results should serve as a model for protein-protein interactions, in general.