ARTICLE pubs.acs.org/JACS Microscopic Analysis of Protein Oxidative Damage: Effect of Carbonylation on Structure, Dynamics, and Aggregability of Villin Headpiece Drazen Petrov and Bojan Zagrovic* Department of Structural and Computational Biology, Max F. Perutz Laboratories, University of Vienna, Campus Vienna Biocenter 5, Vienna AT-1030, Austria Mediterranean Institute for Life Sciences, Mestrovicevo setaliste bb, Split HR-21000, Croatia Department of Physics, Faculty of Science, University of Split, Teslina 12, Split HR-21000, Croatia bS Supporting Information 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. ’ INTRODUCTION Proteins are frequently modiﬁed by diﬀerent reactions invol- ving reactive oxygen species (ROS), including metal-catalyzed carbonylation, oxidation of aromatic and sulfur-containing amino acid residues, oxidation of the protein backbone, or even protein fragmentation due to backbone breakage.1 3 One of the most important mechanisms of oxidative damage to proteins is metal- catalyzed carbonylation, where ROS are created in a Fenton-type reaction involving transition metals such as iron or copper.4 Quantitatively the most important products of carbonylation of amino acids are aminoadipic semialdehyde (Asa), derived from lysine, and glutamic semialdehyde (Gsa), derived from arginine and proline residues (Figure 1a).5,6 Albeit to a much lower extent, carbonyl groups can also be introduced in proteins by direct carbonylation of threonine residues, a secondary reaction with aldehydes produced during lipid peroxidation, or a secondary reaction with carbonyl derivatives generated in reaction of reducing sugars.1,2 As compared to other modiﬁcations induced by ROS, carbonylation is relatively diﬃcult to induce, but once proteins get carbonylated, the change is permanent due to the irreversible nature of the reaction.7,8 For this reason, protein carbonyl content is by far the most widely used marker of protein oxidation.9 The cellular carbonylation level increases with age, with the concen- tration of carbonylated proteins rising exponentially during the last third of the life span in a wide range of organisms.3,10 Furthermore, carbonylation is associated with age-related dis- orders such as neurodegenerative diseases, cancer, and diabetes, but it should be emphasized that it is still unclear whether carbonylation is a direct cause of aging or just a consequence and a useful reporter on aging.4 Importantly, the presence of highly carbonylated protein aggregates has been observed in many of these diseases, but so far no clear causal relationship between carbo- nylation and aggregation has been established.11,12 Finally, the extreme robustness to ionizing radiation and UV light of some extremophile bacteria such as Deinococcus radiodurans has been shown to depend on eﬃcient protection of the proteome against basal and radiation-induced protein carbonylation.13 Received: November 24, 2010 Published: April 20, 2011 r 2011 American Chemical Society 7016 dx.doi.org/10.1021/ja110577e | J. Am. Chem. Soc. 2011, 133, 7016–7024