Structural Characterization of λ-Repressor Folding from All-Atom Molecular Dynamics Simulations

Abstract

The five-helix bundle λ-repressor fragment is a fast-folding protein. A length of 80 amino acid residues puts it on the large end among all known microsecond folders and its size poses a computational challenge for molecular dynamics (MD) studies. We simulated the folding of a novel λ-repressor fast-folding mutant (λ-HG) in explicit solvent using an all-atom description. By means of a recently developed tempering method, we observed reversible folding and unfolding of λ-repressor in a 10-microsecond trajectory. The folding kinetics was also investigated through a set of MD simulations run at different temperatures that together covered more than 125 microseconds. The protein was seen to fold into a native-like topology at intermediate temperature and a slow-folding pathway was identified. The simulations suggest new experimental observables for better monitoring the folding process, and a novel mutation expected to accelerate λ-repressor folding.

Figure 1

C_α -RMSD relative to the 3KZ3 crystal structure (shown at top left) calculated from an enhanced sampling simulation (Sim-ES in Table 1). The C_α -RMSD native value is defined by the mean value (dashed line) from the 300-ns equilibrium simulation of the native structure (Sim-N359 in Table 1). The protein folded reversibly into the native value twice in the simulation. Occupancies of the top 20 clusters throughout the trajectory are shown in red. The representative folded structure, identified through the most populated cluster, is shown at top right. The crystal structure is superimposed as a transparent cartoon representation for comparison. Protein coloring runs blue to red from N-terminus to C-terminus.

Figure 2

Temperature dependence of the histogram distribution of radius of gyration R_gyr and C_α -RMSD values at T = 305 ± 5 K (top) and at T = 595 ± 5 K (bottom). The histogram distribution was determined from simulation Sim-ES and the associated probability, as shown, is normalized such that the highest value is 1.

Figure 3

Characterization of λ-repressor folding trajectory at T = 329 K, T = 359 K, and T = 389 K taken from simulations Sim-F329, Sim-F359, and Sim-F389, respectively. C_α -RMSD values have been calculated relative to the crystal structure 3KZ3; α-content is the fraction of residues that are in α-helical conformation; HPSASA refers to the solvent accessible surface area of hydrophobic groups. The native ranges are defined by the mean value (red solid line) ± standard deviations (green dashed line) from the 300-ns equilibrium simulations of the native structure at the respective temperatures (Sim-N329, Sim-N359, and Sim-N389 in Table 1)

Figure 4

Observables monitored computationally along the trajectory Sim-F359. (a) C_α -RMSD for the first two helices (from residue Gln-9 to Asp-38) relative to the crystal structure. (b) Distance between sidechain of Trp-22 and sidechain of His-33. (c) Solvent accessible surface area of the sidechain of Trp-22 (Trp-SASA). The crystal structure and the partially folded structure reached in the simulation are provided at the top (same color scheme as in Figure 1). The experimental probe residues tryptophan from helix 1 and histidine from helix 2 are shown in licorice representation.