RESUMO
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.
