Probing the Action of Chemical Denaturant on an Intrinsically Disordered Protein by Simulation and Experiment
Alexander V. Grishaev, Wenwei Zheng, Alessandro Borgia, Karin Buholzer, Benjamin Schuler, Robert Best
Chemical denaturants are the most commonly used agent for unfolding proteins, and are thought to act by better solvating the unfolded state. Improved solvation is expected to lead to an expansion of unfolded chains by denaturant, providing a sensitive probe of its action. However, different experiments have so far yielded qualitatively different conclusions regarding this expansion. Here, we use all-atom molecular simulations to investigate the effect of urea and guanidinium chloride on the structure of the intrinsically disordered protein ACTR, which can be studied by experiment over a wide range of denaturant concentration. Using unbiased molecular simulations with a carefully calibrated denaturant model, we find that the protein chain swells with increasing denaturant concentration. Examining the denaturant interactions, we find that this is due to the favorable association of urea with the backbone of all residues and with the side-chains of all residues except for aspartate and glutamate. The urea-water transfer free energies inferred from this association are in reasonable accord with experimental estimates from model compounds. Urea interactions with the backbone are dominated by hydrogen bonding, while interactions with side-chains include other contributions. Our simulations also support some assumptions needed for each experiment to accurately reflect changes in protein size, namely that the commonly used FRET chromophores do not qualitatively alter the results, and that possible effects such as preferential solvent partitioning into the interior of the chain do not interfere with the determination of radius of gyration from the SAXS experiments. Our results demonstrate the power of atomistic simulations with accurately balanced intermolecular interactions for the molecular interpretation of diverse experimental data.