Folding transition-state and denatured-state ensembles of FSD-1 from folding and unfolding simulations

Abstract

Characterization of the folding transition-state ensemble and the denatured-state ensemble is an important step toward a full elucidation of protein folding mechanisms. We report herein an investigation of the free-energy landscape of FSD-1 protein by a total of four sets of folding and unfolding molecular dynamics simulations with explicit solvent. The transition-state ensemble was initially identified from unfolding simulations at 500 K and was verified by simulations at 300 K starting from the ensemble structures. The denatured-state ensemble and the early-stage folding were studied by a combination of unfolding simulations at 500 K and folding simulations at 300 K starting from the extended conformation. A common feature of the transition-state ensemble was the substantial formation of the native secondary structures, including both the alpha-helix and beta-sheet, with partial exposure of the hydrophobic core in the solvent. Both the native and non-native secondary structures were observed in the denatured-state ensemble and early-stage folding, consistent with the smooth experimental melting curve. Interestingly, the contact orders of the transition-state ensemble structures were similar to that of the native structure and were notably lower than those of the compact structures found in early-stage folding, implying that chain and topological entropy might play significant roles in protein folding. Implications for FSD-1 folding mechanisms and the rate-limiting step are discussed. Analyses further revealed interesting non-native interactions in the denatured-state ensemble and early-stage folding and the possibility that destabilization of these interactions could help to enhance the stability and folding rate of the protein.

Figure 1

Native structure of FSD-1. The helix is in red and the β-hairpin is in yellow. The side chains at the helix/sheet interface have been labeled with single letter code and residue index.

Figure 2

The 2-dimensional population distribution around native state (NTRAJ) at 300K. The RMSD and R_g are the reaction coordinates. The population is represented by the color gradient where red is the most populated area.

Figure 3

R_g and RMSD from two representative unfolding trajectories of the UTRAJ set at 500K.

Figure 4

The 2-dimensional population contour from the unfolding trajectories (UTRAJ) at 500K. The coloring scheme is same at Fig 2.

Figure 5

R_g and RMSD of two representative trajectories of the folding simulations (FTRAJ) at 300K.

Figure 6

The 2-dimensional population distribution from the folding trajectories (FTRAJ) at 300K. The coloring scheme is same as Fig 2.

Figure 7

Comparison of C_α-C_α contact maps calculated for the unfolding (UTRAJ, lower-right triangle) and folding (FTRAJ, upperleft triangle) simulations. The gray scale indicates the fractional occupancy. The cutoff distance is 6 Å.

Figure 8

Representative structures of the most populated clusters in folding trajectories (FTRAJ).

Figure 9

Average percentage helix (blue) or β-sheet (black) from the five folding trajectories (FTRAJ). The secondary structures of the native structures are shown in green (β-sheet) and red (helix).

Figure 10

RMSD of four trajectories at 300K started from unfolding-TSE (TSTRAJ). The labels (a)–(d) indicate the corresponding starting structures (a)–(d) shown in Figure 11.

Figure 11

Representative structures of the transition state ensemble that were used as the starting structures in ‘TSTRAJ’ simulations. Close resemblance to the native secondary structures is readily apparent.

Figure 12

C_α-C_α contact map in the native NMR structure (lower-right triangle) and that averaged over TSE structures (upper-left triangle). The cutoff is 6.0 Å.