Journal of the American Chemical Society ARTICLE Figure 1. (a) Chemical structures of Asa, derived by carbonylation of Lys, and Gsa, derived by carbonylation of Arg and Pro. (b) Villin structure. The seven quantitatively most important carbonylable sites are colored as follows: Pro, yellow; Arg, red; Lys, blue. The basal level of carbonylation in cells is approximately 2 nmol of carbonyl per milligram of protein, while the level that appears to correlate with cell and organism death is approximately 6 nmol of carbonyl per milligram of protein, corresponding to about one carbonylated residue per 4000 amino acids on average.3,10,13 Although these average numbers are actually relatively low, it has been speculated that cellular aging is a direct consequence of the loss of structural stability, unfolding, and exposure of hydro- phobic residues of select proteins upon carbonylation.11 However, to the best of our knowledge, only a few high-resolution studies of structural stability of proteins in the face of oxidative stress have been carried out, and not one focusing explicitly on carbonylation. Most studies focused on the gross functional and structural consequences of oxidation, but with typically very little atomistic details.14 20 A common denominator of most of these studies is that oxidation of amino acids leads to local disruption of tertiary structure of proteins with a concomitant exposure of hydrophobic amino acids and subsequent aggregation. Here, we use molecular dynamics (MD)21 simulation to model the carbonylated villin headpiece molecule. Villin is a tissue-speciﬁc actin-binding protein involved in diﬀerent func- tions such as cell motility, deﬁnition of morphology, and cell death,22 and it carries out these functions by bundling, nucleat- ing, capping, and severing actin ﬁlaments.23 Our interest in villin was motivated by two principal rationales. First, oxidative stress in non-muscle mammalian cells is known to cause major changes in cellular morphology and structure of the actin cytoskeleton.24,25 Second, the 36-residue C-terminal headpiece domain of villin is one of the most widely studied and best understood proteins when it comes to folding mechanism and stability.26 33 Here, we study atomistic-level changes in structure and dynamics of the villin headpiece at diﬀerent carbonylation levels. In addition to speciﬁc eﬀects, such as the disruption of a key salt bridge and a proline kink or alteration of the hydrophobic eﬀect, we ask what overall level of carbonylation can be tolerated without major eﬀects on the molecule’s structural and dynamical integrity. Finally, we ask how does carbonylation aﬀects the intrinsic aggregability of the molecule by altering the basic physicochem- ical properties of the aﬀected amino acids. ’ MATERIALS AND METHODS We have used MD to study the stability of the carbonylated villin headpiece domain (sequence: MLSDEDFKAVFGMTRSAFANLPLWK- QQNLKKEKGLF). The seven bolded letters mark the quantitatively most important carbonylable amino acids (K, R, and P) in villin headpiece (Figure 1b). The simulations were run using the Gromacs 3.3.3 biomolecular simulation package. United-atom GROMOS 45A3 force ﬁeld,34,35 SPC explicit water,36 and a 2 fs integration step were used. For electrostatics calculations, reaction ﬁeld was employed with a cutoﬀ of rc = 1.4 nm and the dielectric constant of εrf = 65. An NMR structure of the villin headpiece domain (PDB code 1VII) was used as the starting structure.26 After steepest descent minimization was per- formed in vacuum (500 steps) and subsequently in water (1500 steps), the system was equilibrated by gradually increasing the temperature (from 100 to 300 K) over 100 ps with gradually decreasing position restraints (from 25 000 to 5000 kJ mol 1 nm 2) at constant volume and temperature, and ﬁnally additionally equilibrated for 20 ps at constant pressure and temperature of 1 bar and 300 K. The temperature and pressure in all production simulations were kept at 300 K and 1 bar using a Berendsen thermostat (τT = 0.05 ps) and barostat (τp = 1 ps and compressibility = 4.5 10 5 bar 1).37 Since the villin headpiece domain has seven potential carbonylation sites belonging to the quantitatively most important carbonylable residues K, R, and P (see sequence above), the total number of all possible carbonylation combinations of these residues is 27 = 128. Altogether, 136 independent 110-ns-long trajectories were generated for a total of 14.96 μs of simulation time (ﬁve copies of the native and completely carbony- lated villin headpiece plus one copy of every other combination: 5 þ 5 þ 126 = 136). Coordinates were output every 100 ps, and for the analysis of equilibrium properties, only the last 25 ns of each trajectory was used. Carbonylation modiﬁcations were introduced before energy minimization by changing the villin headpiece coordinate ﬁle and introducing param- eters for the two carbonylated amino acids, Asa and Gsa, in the force ﬁeld. Details of parametrization for Asa and Gsa are given in the Supporting Information (SI). For analysis, the simulations were divided into four sets: (1) simulations in set K include diﬀerent levels of carbonylation of Lys residues, (2) those in set KR include Arg15 carbonylation together with diﬀerent levels of Lys carbonylation, (3) those in set KP include carbonylation of Pro22 together with diﬀerent levels of Lys carbonylation, and (4) those in set KRP include simultaneous Arg15 and Pro22 carbonylation with diﬀerent levels of Lys carbonylation. The trajectories were analyzed primarily using Gromacs tools,38 including calculation of root-mean-square deviation (rmsd), solvent-accessible sur- face area (SASA), molecular volume, and distances between groups of atoms, except for (1) secondary structure analysis, where DSSP39 was used, (2) conformational entropy calculations, where quasiharmonic approximation for calculating entropy was used as described in refs 40 and 41, and (3) characterization of hydrophobic properties of protein surface, where the molecular hydrophobicity potential (MHP) calculation was employed, as described by Efremov et al.42 Throughout the paper, atom-positional rmsd after rotational-translational ﬁtting was calculated with respect to the native NMR villin headpiece structure (PDB code 7017 dx.doi.org/10.1021/ja110577e |J. Am. Chem. Soc. 2011, 133, 7016–7024