Molecular dynamics simulations of protein folding from the transition state

Abstract

Putative transition-state ensemble (TSE) conformations of src SH3 were identified by monitoring the deviation from the experimental phi values along molecular dynamics (MD) simulations of unfolding. Sixty MD trajectories (for a total of about 7 micros) were then started from the putative TSE. About one-half of the 60 runs reached the folded state while unfolding was observed in the remaining half of the runs. This result validates phi-value analysis as an approach to obtain structural information on the transition state. It also demonstrates that an atomic resolution description of the TSE can be extracted from MD simulations. All conformations in the TSE have the central three-stranded beta-sheet formed in agreement with experimental data. An elongation of strand beta 2 as well as non-native side-chain interactions between the diverging turn and the distal hairpin are observed. The simulation results indicate that the tight packing of the side chains between the diverging turn and the distal hairpin is a necessary condition for rapid folding. Contacts between residues in the most structured element of the TSE, the central beta-sheet, are kinetically more important than those between the N- and C-terminal strands.

Figure 1

Different conformational states of src SH3. (a) Stereoview (wall eyes) of the ribbon representation of the crystal structure (27). (b) Stereoview (wall eyes) of six transition-state conformations superimposed to the central β-sheet of the crystal structure. β1 and the RT loop are colored in cyan, the diverging turn and central β-sheet (β2-β4) are in yellow, and the 3₁₀ helix and β5 are in magenta. The N and C termini are indicated by a blue and red sphere, respectively. The native structure is shown in green (thick line). (c) β1-β5-disrupted. (d) β3-β4-disrupted. All figures were prepared with MOLMOL (28).

Figure 2

Properties of the TSE. (a and b) Comparison of φ with φ. (a) The 18 φ used to identify the TSE by the φ-rmsd criterion. (b) Thirty-two residues with well defined φ. (c) C_α rmsd from the native state as a function of residue number. The native secondary structural elements are indicated above the diagram.

Figure 3

Evolution of the C_α rmsd and Q as a function of time in two folding and two unfolding simulations at 315 K started from the same transition-state structure. The dotted lines indicate the threshold values of folding and unfolding.

Figure 4

Folding from the TSE. Snapshots from one of the TS4 folding trajectories at (from left to right and top to bottom) 0, 0.6, 1.0, 2.0, 7.7, and 15.4 ns. The first (Top Left) and last (Bottom Right) conformations have a C_α rmsd from the native structure of 5.8 and 1.9 Å, respectively. In all frames, segments of the polypeptide chain that correspond to the native β-strands and 3₁₀ helix are colored in cyan and red, respectively, whereas loops and segments with nonnative regular secondary structure are in gray.