Understanding folding and design: replica-exchange simulations of "Trp-cage" miniproteins

Abstract

Replica-exchange molecular dynamics simulations in implicit solvent have been carried out to study the folding thermodynamics of a designed 20-residue peptide, or "miniprotein." The simulations in this study used the amber (parm94) force field along with the generalized Born/solvent-accessible surface area implicit solvent model, and they spanned a range of temperatures from 273 to 630 K. Starting from a completely extended initial conformation, simulations of one peptide sequence sample conformations that are <1.0 A Calpha rms positional deviation from structures in the corresponding NMR ensemble. These folded states are thermodynamically stable with a simulated melting temperature of approximately 400 K, and they satisfy the majority of experimentally observed NMR restraints. Simulations of a related mutant peptide show a degenerate ensemble of states at low temperature, in agreement with experimental results.

Fig. 1.

Sample structures from the TC5b (Left) and TC3b (Right) simulations. Simulated structures are shown in green. (Top) Initial extended conformation of each peptide. (Middle) Peptide structures after 50 ps of 298-K molecular dynamics (MD) equilibration. (Bottom) Final 298-K conformation for each peptide after the 4-ns replica-exchange run. The simulated structures are shown superimposed on the first conformation of the TC5b NMR ensemble (shown in red). The structures were superimposed based on C^α RMSD. These images were generated with the pymol program.

Fig. 2.

(A) The fraction of conformations for each peptide that are within 2.0 Å C^α RMSD of any of the 38 structures of the TC5b NMR ensemble as a function of temperature. (B) The fraction of conformations for each peptide that are within 2.0 Å C^α RMSD of the average structure for that peptide from the simulation at 250 K.

Fig. 3.

(A) The fraction of the 169 experimental NMR distance measurements that were violated by the TC5b simulation data as a function of temperature. Similar experimental data were not available for TC3b. (B) Average number of α-helical hydrogen bonds that are present in each simulation as a function of temperature.

Fig. 4.

The fraction of conformations for each peptide that belong to the largest conformational cluster at each temperature. Conformations were clustered by using a simple iterative scheme based on a C^α DME threshold of 1.0 Å.

Fig. 5.

Structures of the major cluster centers from the TC5b and TC3b simulations. Cartoon backbone representations are shown for the centers of the three largest clusters from the 250-K simulations for both peptides. The simulation structures (green, blue, and yellow, in order of cluster size) are shown superimposed on the first structure from the TC5b NMR ensemble (red) based on the C^α positions of residues 2–9. (A) Cluster centers for TC5b, where the top three clusters all have very similar conformations. These clusters account for 87%, 6%, and 5% of the sampled TC5b conformations at 250 K. (B) The center of the largest TC3b 250-K cluster in green (37% of sampled conformations). (C) The second largest TC3b 250-K cluster in blue (21% of sampled conformations). (D) The third largest TC3b cluster in yellow (19% of sampled conformations). (B–D) The three major TC3b clusters differ significantly in the packing of the C-terminal polyproline helix against the N-terminal α-helix. These images were generated with the pymol program.

Fig. 6.

The heat capacity of each simulated peptide system is shown as a function of temperature. The TC5b simulation data points are shown connected with a solid line, whereas the TC3b simulation data points are connected by a dashed line. Error bars represent one standard deviation about the mean.