Journal of the American Chemical Society ARTICLE Figure 3. Structural stability of the villin headpiece as a function of carbonylation level: (a) rmsd from the native villin headpiece structure 1VII, (b) sum of the distances between the three core phenylalanines, (c) number of residues in R-helical conformation, and (d) quasi-harmonic conformational entropy normalized by number of atoms, relative to that of native villin headpiece, all shown as a function of the number of carbonylated residues. Each line is obtained from a diﬀerent subset of simulations: the lightest lines (9), set K; dark gray lines (2), set KR; light gray lines ([), set KP; the darkest lines (b), set KRP. All points in the plots were calculated as averages over the last 25 ns in each simulation and all simulations in a given subset with the same number of carbonylated residues and are shown with standard deviations. Red lines represent averages of given observables over the ﬁve native simulations, increased by the standard deviations, and were used as a criterion for determining whether a given structure is folded or unfolded. core of the protein, keeping its R-helices together,32 and (2) a salt bridge between Asp4 and Arg15 residues.33 Simultaneous carbo- nylation of all seven major carbonylable residues in the protein results in the loss of approximately 40% of its R-helical secondary structure and most of its tertiary structure in approximately 100 ns (Figure 2a,b). While the simulated native protein keeps its R-helical content and tertiary structure largely intact over the same period, the carbonylated protein unfolds and starts explor- ing the accessible areas of the Ramachandran map more freely. Unfolding of the protein upon carbonylation occurs simulta- neously with the disintegration of its Phe core (Phe7, Phe11, and Phe18) (Figure 2c,d). Interestingly, the solvent exposure of the Phe core increases multiple-fold as a consequence of carbonyla- tion (Figure 2c), while the total SASA of the protein remains the same although the core residues become exposed to the surface. What happens if just a subset of diﬀerent carbonylable residues in villin headpiece are carbonylated, and how does this depend on the type and position of the residues aﬀected? To address this, we have divided our simulations into four distinct sets which were analyzed separately (Table 1). The four sets were organized in such a way to probe diﬀerent structural eﬀects of carbonylation: surface hydrophobicity change, Asp4- Arg15 salt bridge removal, proline kink removal, or a combina- tion thereof. Interestingly, the structure remains intact when it comes to tertiary fold (as measured by rmsd from the native structure), core compactness (as measured by the sum of distances between the centers of mass of core phenylalanines, ∑dPhe), and secondary structure (as measured by the number of R-helical residues) for all single and double carbonylation hits, regardless of their type or location (Figure 3a c). More- over, the structural features remain largely unchanged with regard to all structural measures, even with all ﬁve lysine residues carbonylated (K set) (Figure 3a c). This is striking as Asa, the product of carbonylation of Lys residues, is Figure 4. Pairwise rmsd density plot. Each point represents the results of comparison of two ensembles of structures from a given pair of simulations of given combinations of carbonylated residues (linear average of the distribution of all-against-all rmsd evaluations for the two ensembles). Altogether 128 combinations were analyzed, and both x and y axis range from 0 to 7 (number of carbonylated residues), where 0 represents the native ensemble and 7 represents the fully carbonylated ensemble. Backbone atoms were used for alignment and rmsd calculations. signiﬁcantly less hydrophilic than Lys itself (i.e., its hydration free energy is more positive, SI Figure S1), which could lead to a signiﬁcant disruption of the stabilizing hydrophobic eﬀect. Our thermodynamic integration (TI) calculations suggest that ΔGhydr between them exceeds 10 kJ mol 1 in uncharged form and 230 kJ mol 1 in charged form (SI Figure S1), with the carbonylation of Arg having a similar eﬀect. 7019 dx.doi.org/10.1021/ja110577e |J. Am. Chem. Soc. 2011, 133, 7016–7024