Journal of the American Chemical Society ARTICLE Figure 2. Complete carbonylation leads to unfolding of the villin headpiece. (a) All-atom rmsd from the native villin headpiece structure 1VII (residues 43 74) as a function of time. (b) The number of residues in R-helical conformation as a function of time. In both plots the solid line (9) represents native villin headpiece while the dashed line (b) represents fully carbonylated villin headpiece simulations. (c) Total SASA of the three core Phe residues normalized by the total SASA of fully solvent-exposed Phe. Empty bars represent native villin headpiece while gray bars represent fully carbonylated villin headpiece simulations. All curves and bars in panels a c were obtained as averages over ﬁve independent native/fully carbonylated simulations and are shown with standard deviations. (d) A representative structure of native (left) and completely carbonylated (right) villin headpiece showing the surface of the three core phenylalanines in red and the rest of the protein in white. Table 1. Types of Trajectories Studieda set K KR KP traj no. 36 32 32 carb res 0 5 Lys 0 5 Lys þ Arg 0 5 Lys þ Pro evaluated eﬀects Δ hydrophobicity salt bridge disruption þ Δ hydrophobicity proline kink disruption þ Δ hydrophobicity all three eﬀects combined KRP 36 0 5 Lys þ Arg þ Pro a Each set contains a given carbonylation event plus every possible combination of carbonylating the 0 5 Lys residues in the villin headpiece. Set K includes ﬁve simulations of the native villin headpiece, i.e., a combination with no Lys residues carbonylated. Similarly, set KRP contains ﬁve simulations of the completely carbonylated villin headpiece, i.e., a combination with all ﬁve Lys residues carbonylated. 1VII, residues 43 74; the two residues at each end of the peptide were excluded in calculation as they tend to be dynamic). The PDB structure 1VII starts with the residue 41 and ends with the residue 76, since the complete villin headpiece is 76 residues long, while the reported structure captures just the 36-residues-long C-terminal domain. We used 1 36 numbering here, using backbone atoms for alignment and all atoms for rmsd calculation, if not stated otherwise. Exposure of residues to water was estimated by calculating solvent accessibility fraction, calculated as a fraction of SASA calculated for a given side chain in the context of the villin headpiece structure as compared with the SASA of the same side chain when completely exposed to solvent. Distance between given residues was calculated as the distance between centers of masses of their side chains. We used the formula proposed by Chiti et al.43 to calculate the change in aggregability upon carbonylation (for structurally destabilized proteins): lnðνmut=νwtÞ ¼ AΔHydr þ B ΔΔGcoil-R þ ΔΔGβ-coilÞ þ CΔCharge ð where νmut and νwt are rates of forming aggregates, ΔHydr is the change in hydrophobicity according to the hydrophobicity scale based on water-to-octanol partition,43 ΔΔGcoil-R þ ΔΔGβ-coil is the change in propensity to form an R-helix over a β-sheet, ΔCharge is the change in the absolute value of protein net charge, and A = 0.633, B = 0.198, and C = 0.491 are ﬁtted constants. We used only the ﬁrst term and the last term, with water/octanol partitioning hydrophobicity values for Asa and Gsa obtained from the MHP calculation described by Efremov et al.42 and the correlation given in Figure 5a (below), but also provided upper and lower bounds derived from the standard deviation of R-helix and β-sheet propensities over the 27 protein studied by Chiti et al.43 We used the “Zyggregator” 44 model to study the carbonylation-induced change in intrinsic aggregability directly from the native state of the villin headpiece. For this calculation, Asa and Gsa residues were replaced by amino acids that most closely match them in terms of charge and MHP, i.e., Leu and Val, respectively. We could not use Asa and Gsa directly, as not all parameters (such as R and β propensities) needed for prediction are available at this point. 1 ð Þ ’ RESULTS Effect of Carbonylation on Protein Structural and Dyna- mical Integrity. The structure of the villin headpiece domain is principally stabilized by (1) three phenylalanines buried in the 7018 dx.doi.org/10.1021/ja110577e |J. Am. Chem. Soc. 2011, 133, 7016–7024