Protein phosphorylation is a crucial process in various cellular functions, and its irregularities have been implicated in several diseases, including cancer. Antibodies are commonly employed to detect protein phosphorylation in research. However, unlike the extensive studies on recognition mechanisms of the phosphate group by proteins such as kinases and phosphatases, only a few studies have explored antibody mechanisms. In this study, we produced and characterized two rabbit monoclonal antibodies that recognize a mono-phosphorylated Akt peptide. Through crystallography, thermodynamic mutational analyses, and molecular dynamics simulations, we investigated the unique recognition mechanism that enables higher binding affinity and selectivity of the antibodies compared to other generic proteins with lower binding affinity to phosphorylated epitopes. Our results demonstrate that molecular dynamics simulations provide novel insights into the dynamic aspects of molecular recognition of post-translational modifications by proteins beyond static crystal structures, highlighting how specific atomic level interactions drive the exceptional affinity and selectivity of antibodies.

中文翻译：

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揭示磷酸化表位特异性兔抗体中高亲和力和选择性背后的结构机制。


蛋白质磷酸化是各种细胞功能的关键过程，其不规则性与包括癌症在内的多种疾病有关。抗体通常用于检测研究中的蛋白质磷酸化。然而，与激酶和磷酸酶等蛋白质对磷酸基团识别机制的广泛研究不同，只有少数研究探讨了抗体机制。在这项研究中，我们生产并表征了两种识别单磷酸化 Akt 肽的兔单克隆抗体。通过晶体学、热力学突变分析和分子动力学模拟，我们研究了独特的识别机制，与对磷酸化表位结合亲和力较低的其他通用蛋白相比，该机制使抗体具有更高的结合亲和力和选择性。我们的结果表明，分子动力学模拟为静态晶体结构之外蛋白质对翻译后修饰的分子识别的动力学方面提供了新的见解，突出了特定的原子水平相互作用如何驱动抗体的特殊亲和力和选择性。