Post-translational modifications of proteins expand their functional diversity, regulating the response of cells to a variety of stimuli. Among these modifications, phosphorylation is the most ubiquitous and plays a prominent role in cell signaling. The addition of a phosphate often affects the function of a protein by altering its structure and dynamics. However, these alterations are often difficult to study and the functional and structural implications remain unresolved. New approaches are emerging to overcome common obstacles related to the production and manipulation of these samples. Here, we summarize the available methods for phosphoprotein purification and phosphomimetic engineering, highlighting the advantages and disadvantages of each. We propose a general workflow for protein phosphorylation analysis combining computational and biochemical approaches, building on recent advances that enable user-friendly and easy-to-access Molecular Dynamics simulations. We hope this innovative workflow will inform the best experimental approach to explore such post-translational modifications. We have applied this workflow to two different human protein models: the hemeprotein cytochrome c and the RNA binding protein HuR. Our results illustrate the usefulness of Molecular Dynamics as a decision-making tool to design the most appropriate phosphomimetic variant.

蛋白质的翻译后修饰可扩展其功能多样性，从而调节细胞对多种刺激的反应。在这些修饰中，磷酸化是最普遍的，并且在细胞信号转导中起重要作用。磷酸盐的添加通常会通过改变蛋白质的结构和动力学来影响其功能。但是，这些更改通常很难研究，功能和结构方面的影响仍未解决。出现了新的方法来克服与这些样品的生产和操作有关的常见障碍。在这里，我们总结了可用于磷蛋白纯化和模拟磷酸化工程的方法，重点介绍了每种方法的优缺点。我们基于结合了用户友好和易于访问的分子动力学模拟的最新进展，提出了一种将计算和生化方法相结合的蛋白质磷酸化分析的一般工作流程。我们希望这种创新的工作流程将为探索此类翻译后修饰的最佳实验方法提供参考。我们已将此工作流程应用于两种不同的人类蛋白质模型：血红蛋白细胞色素c和RNA结合蛋白HuR。我们的结果说明了分子动力学作为设计最合适的拟磷酸化变体的决策工具的有用性。