The folding transition-state ensemble of a four-helix bundle protein: helix propensity as a determinant and macromolecular crowding as a probe

Abstract

The four-helix bundle protein Rd-apocyt b(562), a redesigned stable variant of apocytochrome b(562), exhibits two-state folding kinetics. Its transition-state ensemble has been characterized by Phi-value analysis. To elucidate the molecular basis of the transition-state ensemble, we have carried out high-temperature molecular dynamics simulations of the unfolding process. In six parallel simulations, unfolding started with the melting of helix I and the C-terminal half of helix IV, and followed by helix III, the N-terminal half of helix IV and helix II. This ordered melting of the helices is consistent with the conclusion from native-state hydrogen exchange, and can be rationalized by differences in intrinsic helix propensity. Guided by experimental Phi-values, a putative transition-state ensemble was extracted from the simulations. The residue helical probabilities of this transition-state ensemble show good correlation with the Phi-values. To further validate the putative transition-state ensemble, the effect of macromolecular crowding on the relative stability between the unfolded ensemble and the transition-state ensemble was calculated. The resulting effect of crowding on the folding kinetics agrees well with experimental observations. This study shows that molecular dynamics simulations combined with calculation of crowding effects provide an avenue for characterize the transition-state ensemble in atomic details.

Figure 1

Structure of the four-helix bundle protein Rd-apocyt b₅₆₂. In the color version of this figure, helices I, II, III, IV-N, and IV-C are displayed in red, blue, cyan, yellow, and green, respectively.

Figure 2

Root mean-square deviations of the simulations from the starting NMR structure, calculated over C_α atoms. The curves are smoothed using the bezier option in gnuplot. The RMSD of the native-state simulation remains at its low values all the way to the end of the 31-ns trajectory.

Figure 3

Helical status of the individual residues along the six high-temperature trajectories. Each helical residue is indicated by a vertical bar; the bars of the different helical segments have the same color code as in Fig. 1.

Figure 4

Composite profile of helical fractions calculated over the course of five high-temperature simulations. The areas above the individual helices of the native structure are separately shaded.

Figure 5

Intrinsic helix propensities predicted from the Rd-apocyt b₅₆₂ sequence by the AGADIR program. Parameters used in the predictions were: 274 K; 0 ion strength; and pH 7. The areas above the individual helices of the native structure are separately shaded.

Figure 6

Comparison residue helix probabilities of the transition-state ensemble extracted from the simulations and Φ-values (8,16).

Figure 7

Structure of a representative of the transition-state ensemble.

Figure 8

Effects of crowding on the folding and unfolding activation free energies, ΔΔG^‡_TS-U and ΔΔG^‡_TS-N, in units of k_BT where k_B is Boltzmann constant and T is the absolute temperature. Results for three crowder radii (with values shown) are displayed.