Energetic frustration of apomyoglobin folding: role of the B helix

Abstract

Apomyoglobin folds by a sequential mechanism in which the A, G, and H helix regions undergo rapid collapse to form a compact intermediate onto which the central portion of the B helix subsequently docks. To investigate the factors that frustrate folding, we have made mutations in the N-terminus of the B helix to stabilize helical structure (in the mutant G23A/G25A) and to promote native-like hydrophobic packing interactions with helix G (in the mutant H24L/H119F). The kinetic and equilibrium intermediates of G23A/G25A and H24L/H119F were studied by hydrogen exchange pulse labeling and interrupted hydrogen/deuterium exchange combined with NMR. For both mutants, stabilization of helical structure in the N-terminal region of the B helix is confirmed by increased exchange protection in the equilibrium molten globule states near pH 4. Increased protection is also observed in the GH turn region in the G23A/G25A mutant, suggesting that stabilization of the B helix facilitates native-like interactions with the C-terminal region of helix G. These interactions are further enhanced in H24L/H119F. The kinetic burst phase intermediates of both mutants show increased protection, relative to wild-type protein, of amides in the N-terminus of the B helix and in part of the E helix. Stabilization of the E helix in the intermediate is attributed to direct interactions between E helix residues and the newly stabilized N-terminus of helix B. Stabilization of native packing between the B and G helices in H24L/H119F also favors formation of native-like interactions in the GH turn and between the G and H helices in the ensemble of burst phase intermediates. We conclude that instability at the N-terminus of the B helix of apomyoglobin contributes to the energetic frustration of folding by preventing docking and stabilization of the E helix.

Figure 1

pH-dependent unfolding of apomyoglobin and mutants. A. helical content monitored at 222 nm in the CD spectrum. B. fluorescence emission intensity recorded with the excitation wavelength at 288 nm for wild-type (black), G23A/G25A (red), and H24L/H119F (blue). Solid curves were fitted to the data by using linear least-squares algorithm.

Figure 2

Protection factor as a function of residue number for the equilibrium (pH ~4) intermediates. A. wild-type, pH 4 (black), showing the mutation sites for the G23A/G25A mutant protein (red arrows) and the H24L/H119F mutant protein (blue arrows). The location of the helices in the wild-type protein in the fully-folded state are shown at the top of the panel, and their locations in relation to the protection factor data are shown as gray boxes. The position of the G23 and G25 within the B helix and their apparent effect on the protection factor of the wild-type protein at pH 4 is emphasized with a pink box. B. Superposition of the wild-type data from part A (black) with that for the G23A/G25A mutant protein, pH 4 (red). Residues showing a significant change in the mutant are labeled and the extent of the change indicated by arrows, red for increased protection in the mutant and black for decreased protection. C. Superposition of the wild-type data from part A (black) with that for the H24L/H119F mutant protein, pH 3.4 (blue). Residues showing a significant change in the mutant are labeled and the extent of the change indicated by arrows, blue for increased protection in the mutant and black for decreased protection. A significant change is defined as value of > 2 or < 0.5 in the ratio of the protection factors of mutant and wild type at that residue.

Figure 3

Time course of amide protection for the B-helix in A. wild type, B. G23A/G25A, and C. H24L/H119F. A transition curve of the proton occupancy has been fitted by using linear least-squares algorithm for residues whose proton occupancy at 6.4 ms is below 0.8.

Figure 4

Proton occupancy (A0) for the burst phase intermediate as a function of residue number. A. wild type (black) showing the mutation sites for the G23A/G25A mutant protein (red arrows) and the H24L/H119F mutant protein (blue arrows). B. Superposition of the wild-type data from part A (black) with that for the G23A/G25A mutant protein. Residues showing a significant change in the mutant are labeled and the extent of the change indicated by arrows, red for increased protection in the mutant and black for decreased protection. C. Superposition of the wild-type data from part A (black) with that for the H24L/H119F mutant protein. Residues showing a significant change in the mutant are labeled and the extent of the change indicated by arrows, blue for increased protection in the mutant and black for decreased protection. A significant change is defined as dA0 (A0_mutant – A0_{wild type}) greater than 0.1 or less than −0.1.

Figure 5

Differences in the folding behavior of the G23A/G25A and H24L/H119F mutants. A. difference in values of the proton occupancy A0 between wild type and mutants (G23A/G25A, red, H24L/H119F blue). Values greater than 0.1 or less than −0.1 (dotted horizontal lines) for both mutants are circled in black (with vertical black lines connecting the corresponding values for the two mutants) or for the H24L/H119F mutant alone in blue. B. ratio of the protection factor between wild type and mutant (G23A/G25A, red H24L/H119F blue). Values greater than 2 or less than 0.5 (dotted lines) for both mutants are circled in black (with vertical black lines connecting the corresponding values for the two mutants) or for the H24L/H119F mutant alone in blue.

Figure 6

Mapping of the locations of significantly stabilized (blue) or destabilized (red) amides in the kinetic and equilibrium intermediates of the G23A/G25A and H24L/H119F mutants onto the backbone of the X-ray crystal structure of MbCO. Helices A-H are denoted by colored backbone: blue (A), turquoise (B), aquamarine (C), light green (D), green (E), gray (F), yellow (G) and coral (H). A. kinetic intermediate of G23A/G25A. The mutation sites are indicated with pink spheres at the CA positions of G23 and G25. B. kinetic intermediate of H24L/H119F. The locations of the two histidine side chains are indicated as pink spheres. C. equilibrium intermediate of G23A/G25A. D. equilibrium intermediate of H24L/H119F. Mutation sites indicated as in parts A and B. These figures were generated in MolMol.