Journal of the American Chemical Society “mutations” are ln(νmut/νwt) = 3.37 ( 0.73 (Lys to Asa) and ln(νmut/νwt) = 3.85 ( 0.73 (Arg to Gsa), i.e., more than a 30- to 40-fold increase in the aggregation rate for a single carbonylation event, with the error bounds capturing the expected eﬀects of the change in the intrinsic R-helix and β-sheet propensity, not modeled here. Note that this model is sequence-independent and that these changes in aggregability are applicable not just to the villin headpiece but rather to any given positively charged protein. Carbonylation increases aggregability of negatively charged pro- teins as well, but to a somewhat lower extent (ln(νmut/νwt) = 2.39 ( 0.73 for Lys to Asa, and ln(νmut/νwt) = 2.87 ( 0.73 for Arg to Gsa), which is still extremely signiﬁcant. In Figure 7a, we compare this change in aggregability with the predicted change in aggregation rate for 27 diﬀerent point mutations in seven diﬀerent unstructured peptides or intrinsically disordered proteins causing diabetes and diﬀerent neurodegenerative diseases (amylin, two prion peptides, R-synuclein, amyloid β-peptide, tau protein, and a model protein) examined by Chiti et al.43 Note that the latter predictions correlate well with experimental measurements (Pearson r = 0.85). Strikingly, the eﬀect of carbonylation of a single Lys or Arg residue is comparable in magnitude with the most drastic aggregation-inducing mutations in these proteins, with only 2 out of 27 such mutations having a greater eﬀect on protein aggregability than either one of these carbonylation- induced conversions. Finally, to probe the eﬀect of carbonylation on protein aggreg- ability directly from the native state, we have used the model of Vendruscolo et al.,44 replacing Asa and Gsa with amino acids that most closely match them in terms of charge and MHP hydropho- bicity, i.e., Leu and Val, respectively. These approximations notwithstanding, it appears that carbonylation of a single residue indeed signiﬁcantly increases intrinsic aggregability of villin head- piece even in the absence of any major unfolding (Figure 7b). For example, the carbonylation of Pro22 increases its Zagg score, i.e., the aggregability of the region around this residue, from an unfavorable 1 to a highly favorable 0.5. In this model, the Zagg score reﬂects the combined eﬀect of the intrinsic aggregation propensity of a given sequence and its tendency to be structurally unstable in the native state. Our analysis (Figure 7b and SI Figure S3), showing a dramatic increase in Zagg upon carbonylation throughout the villin sequence, suggests that both of these factors change signiﬁ- cantly upon carbonylation in the direction that favors aggregation. ’ DISCUSSION This study presents the ﬁrst-ever computer simulation eﬀort to analyze the eﬀects of carbonylation on protein structure. We have shown that the structure of the completely carbonylated villin headpiece domain unfolds in the course of simulated trajectories. Further analysis showed that extent of destabiliza- tion is site-speciﬁc, and that the disruption of both speciﬁc stabilizing elements (salt bridges and proline kinks) together with surface hydrophobicity change upon carbonylation of a large number of residues is required for protein unfolding. Note, however, that the eﬀect of surface hydrophobicity change cannot be disentangled from other speciﬁc eﬀects: for example, the carbonylation event, which disrupts salt-bridge formation, in- variably also changes the hydrophobicity of the involved residue. Carbonylation of the proline residue is the only case where such a binary eﬀect is avoided, as the native and the carbonylated forms of the residue exhibit similar levels of hydrophobicity. ARTICLE The level of carbonylation at which the villin headpiece, a marginally stable protein,46 unfolds in our simulations (ca. 14% of all residues) is ∼3 orders of magnitude greater than the average level of carbonylation which correlates with cellular senescence and death in vivo (ca. 0.025% of all residues).3,10,13 This suggests that typical cellular levels of carbonylation are not likely to disrupt protein structure, which is not in contradiction with experimental studies.16,18 If carbonylation does not induce major structural destabilization of a typical protein and thus lead to aggregation, as widely assumed,17,47 how does it then promote the formation of cytotoxic aggregates? First, several studies suggest that some proteins are more susceptible to carbonylation than others,9,48 50 which may result in a situation where these proteins are completely carbonylated, while the majority of other proteins are still intact, in agreement with our results. Second, newly synthesized proteins in an old organism might be mis- folded or unfolded due to, for example, accumulated DNA mutations and would therefore be more susceptible to carbony- lation, as has been shown by Dukan et al.51 Finally, modiﬁcations of speciﬁc, functionally important residues may abolish the function of a protein without destabilizing its structure. We propose two novel possibilities to explain this. First, our results suggest that carbonylation just by itself may increase protein aggregability of unstructured polypeptides (Figure 7a). In this scenario, protein unfolding arises because of either carbonylation or some other environmental factors, but impor- tantly, carbonylation makes the unfolded molecule additionally aggregation-prone. Second, our results suggest that carbonyla- tion may increase protein aggregability even in the absence of major unfolding (Figure 7b). From lysozyme to superoxide dismutase to prolactin, there are numerous examples of proteins which undergo amyloid formation under native conditions and without any major unfolding, simply as a consequence of local thermal ﬂuctuations.52 Increasing the local propensity to aggregate through carbonylation may speed this process up. Using TI and MHP approaches, we have shown that the products of carbonylation of Lys and Arg residues are signiﬁcantly more hydrophobic than these amino acids. Such an increase in hydrophobicity, together with a concomitant charge removal, signiﬁcantly increases the intrinsic aggregability of proteins.43 45 On the other hand, the aggregation propensity of proline residues is decreased by precluding β-sheet formation, and their mutations are known to lead to protein aggregation.53 Finally, recent ﬁndings that the three quantitatively most important carbonylable residues (Arg, Lys, and Pro) in many proteins actually serve as key gatekeepers that ﬂank aggregation-prone regions of proteins and prevent aggregation further support our suggestions.54 In general, the list of amyloidogenic, deposition diseases shows a strong overlap with the list of diseases in which high levels of protein carbonylation are detected, including Alzehei- mer’s disease, Parkinson’s disease, diabetes, rheumatoid arthritis, dementia with Lewy bodies, familial amyotrophic lateral sclero- sis, and others.7,9,52,55 Because of the complex pathophysiology of these diseases, the causal link between carbonylation and aggregation is not simple and is still to be fully explored. In fact, not all aggregation-prone proteins associated with these diseases are at the same time highly carbonylable and vice versa. However, there are a number of important examples where this is precisely so. For example, ubiquitin C-terminal hydrolase L1 (UCH-L1), which features critically in sporadic variants of Alzheimer’s and Parkinson’s diseases, forms aggregates but is also highly 7022 dx.doi.org/10.1021/ja110577e |J. Am. Chem. Soc. 2011, 133, 7016–7024