Microscopic Analysis of Protein Oxidative Damage: Effect of Carbonylation on Structure, Dynamics, and Aggregability of Villin Headpiece

Abstract

One of the most important irreversible oxidative modifications of proteins is carbonylation, the process of introducing a carbonyl group in reaction with reactive oxygen species. Notably, carbonylation increases with the age of cells and is associated with the formation of intracellular protein aggregates and the pathogenesis of age-related disorders such as neurodegenerative diseases and cancer. However, it is still largely unclear how carbonylation affects protein structure, dynamics, and aggregability at the atomic level. Here, we use classical molecular dynamics simulations to study structure and dynamics of the carbonylated headpiece domain of villin, a key actin-organizing protein. We perform an exhaustive set of molecular dynamics simulations of a native villin headpiece together with every possible combination of carbonylated versions of its seven lysine, arginine, and proline residues, quantitatively the most important carbonylable amino acids. Surprisingly, our results suggest that high levels of carbonylation, far above those associated with cell death in vivo, may be required to destabilize and unfold protein structure through the disruption of specific stabilizing elements, such as salt bridges or proline kinks, or tampering with the hydrophobic effect. On the other hand, by using thermodynamic integration and molecular hydrophobicity potential approaches, we quantitatively show that carbonylation of hydrophilic lysine and arginine residues is equivalent to introducing hydrophobic, charge-neutral mutations in their place, and, by comparison with experimental results, we demonstrate that this by itself significantly increases the intrinsic aggregation propensity of both structured, native proteins and their unfolded states. Finally, our results provide a foundation for a novel experimental strategy to study the effects of carbonylation on protein structure, dynamics, and aggregability using site-directed mutagenesis.

Modular Organization of α-Toxins from Scorpion Venom Mirrors Domain Structure of Their Targets, Sodium Channels

“…For example, calculation of hydrophobic "complementarity" in the binding pockets at the protein/ligand interface uncovers important principles in molecular recognition, and this idea may be applied to improve docking results (15,28). Moreover, surface hydrophobicity is a good measure for conformational transitions (42,43) and generally "tags" areas of intermolecular recognition (16).…”

“…It represents a powerful tool to calculate spatial distribution of hydrophobic/hydrophilic properties proven to be important in molecular recognition (15). Similar dynamic analysis of the surface hydrophobicity has recently been used in characterization of posttranslational modification effects in globular proteins (42,43).…”

Happily (n)ever after: Aging in the context of oxidative stress, proteostasis loss and cellular senescence

“…In fact, we have shown that upon oxidative stress, part of carbonylated actin is degraded by the proteasome, but depending on the intensity or duration of stimulus, carbonylated actin can form protein aggregates that inhibit the proteasome [80], [94]. Oxidative modifications in proteins such as protein carbonyls, increase the surface hydrophobicity of proteins, dramatically [95]. Carbonylated proteins are more prone to unfold and expose their hydrophobic core, usually concealed inside a folded native protein.…”

“…Carbonylated proteins are more prone to unfold and expose their hydrophobic core, usually concealed inside a folded native protein. At this point, oxidized proteins can interact amongst each other, contributing to an increase in insoluble protein aggregates [76], [95], [96], [97]. The formation of a Schiff-base, resulting from the reaction of a carbonyl group of a carbonylated protein with an amino group of another protein, can further contribute to aggregate formation.…”

Role of Spermidine in Overwintering of Cyanobacteria

“…Only a portion of protein molecules are carbonylated (0.05 to 0.4 carbonyl per 50-kDa protein) in cells (47), and the carbonylation appears to prefer some proteins over others (48)(49)(50). Oxidative damage could inhibit protein functions by modifying critical sites, destabilizing tertiary structure and promoting protein aggregation (51,52). Due to its irreversible nature, carbonylation is widely used to indicate the degree of protein oxidation.…”