Complex Folding Landscape of Apomyoglobin at Acidic pH Revealed by Ultrafast Kinetic Analysis of Core Mutants

Abstract

Under mildly acidic conditions (pH 4-4.5) apomyoglobin (apoMb) adopts a partially structured equilibrium state ( M-state) that structurally resembles a kinetic intermediate encountered at a late stage of folding to the native structure at neutral pH. We have previously reported that the M-state is formed rapidly (<1 ms) via a multistate process and thus offers a unique opportunity for exploring early stages of folding by both experimental and computational techniques. In order to gain structural insight into intermediates and barriers at the residue level, we studied the folding/unfolding kinetics of 12 apoMb mutants at pH 4.2 using fluorescence-detected ultrafast mixing techniques. Global analysis of the submillisecond folding/unfolding kinetics vs urea concentration for each variant, based on a sequential four-state mechanism ( U ⇔ I ⇔ L ⇔ M), allowed us to determine elementary rate constants and their dependence on urea concentration for most transitions. Comparison of the free energy diagrams constructed from the kinetic data of the mutants with that of wild-type apoMb yielded quantitative information on the effects of mutations on the free energy (ΔΔ G) of both intermediates and the first two kinetic barriers encountered during folding. Truncation of conserved aliphatic side chains on helices A, G, and H gives rise to a stepwise increase in ΔΔ G as the protein advances from U toward M, consistent with progressive stabilization of native-like contacts within the primary core of apoMb. Helix-helix contacts in the primary core contribute little to the first folding barrier ( U ⇔ I) and thus are not required for folding initiation but are critical for the stability of the late intermediate, L, and the M-state. Alanine substitution of hydrophobic residues at more peripheral helix-helix contact sites of the native structure, which are still absent or unstable in the M-state, shows both positive (destabilizing) and negative (stabilizing) ΔΔ G, indicating that non-native contacts are formed initially and weakened or lost as a result of subsequent structural rearrangement steps.

Figure 1.

Structure of sperm whale myoglobin (PDB 2JHO) with the heme group removed. The side chains that were subjected to Ala-substitution in this study are shown in the ball-and-stick representation.

Figure 2.

Summary of mutational perturbations in stability (A) and β_L-value (B) obtained by equilibrium fluorescence and CD measurements vs. urea concentration. Red bars in panel A show the unfolding free energy of the M-state, and gray bars shown that of the L-state.

Figure 3.

The folding kinetics of V10A variant at pH 4.2. Panels A and B show the refolding and unfolding kinetic traces at various urea concentrations monitored by Trp fluorescence, respectively. The solid lines represent the time course of folding (A) or unfolding (B) predicted by global fitting of a four-state mechanism (Scheme 1) to the combined kinetic data. Panel C shows the corresponding chevron plots. Open circles were obtained by individual fitting of kinetic traces to single or double exponential functions. The rates assigned to λ₁, λ₂ and λ₃ are shown in red, green and blue, respectively. Solid lines represent simulated chevron plots obtained by the global fitting of the combined kinetic and equilibrium data. The corresponding elementary rate constants are shown in the dashed lines. Black lines represent the fitted rate constants of WT apoMb. Panel D shows the free energy diagrams for V10A (red) and WT apoMb (black) derived from the elementary rate constants in the absence of urea.

Figure 4.

The folding kinetics of L76A variant at pH 4.2. As in Figure 3, panels A and B show the refolding and unfolding kinetic traces vs. urea concentration, and panels C and D show the corresponding chevron plot and free energy diagrams, respectively.

Figure 5.

Free energy diagrams (A-D) of apoMb mutants at pH 4.2 in the absence of urea and the free energy perturbations, ΔΔG of each mutant relative to WT apoMb (E-H). The activation free energy for each transition is calculated from the elementary rate constants shown in Table 2, using an Arrhenius pre-exponential factor of 5.0 × 10⁵ s⁻¹. Except for V10A, L69A, L76A and WT, the values for TS3 are shown in dotted lines because a fast unfolding phase (λ₂) that defines this barrier was not observed for these mutants.

Figure 6.

Summary of mutational perturbations in kinetic parameters, including ΔΔG values for all variants studied (A) and Φ-values of primary core mutants (B-D). In panel A ΔΔG values of TS1 are shown in blue, I-state in cyan, TS2 in green, L-state in orange, and M-state in red (see inset). For V10A, L69A and L76A, ΔΔG values of TS3 are shown in gray, while for the other mutants, ΔΔG values of TS3 are not shown.