Probing the Non-Native H Helix Translocation in Apomyoglobin
Folding Intermediates
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Abstract
Apomyoglobin folds via sequential
helical intermediates that are
formed by rapid collapse of the A, B, G, and H helix regions. An equilibrium
molten globule with a similar structure is formed near pH 4. Previous
studies suggested that the folding intermediates are kinetically trapped
states in which folding is impeded by non-native packing of the G
and H helices. Fluorescence spectra of mutant proteins in which cysteine
residues were introduced at several positions in the G and H helices
show differential quenching of W14 fluorescence, providing direct
evidence of translocation of the H helix relative to helices A and
G in both the kinetic and equilibrium intermediates. Förster
resonance energy transfer measurements show that a 5-({2-[(acetyl)amino]ethyl}amino)naphthalene-1-sulfonic
acid acceptor coupled to K140C (helix H) is closer to Trp14 (helix
A) in the equilibrium molten globule than in the native state, by
a distance that is consistent with sliding of the H helix in an N-terminal
direction by approximately one helical turn. Formation of an S108C–L135C
disulfide prevents H helix translocation in the equilibrium molten
globule by locking the G and H helices into their native register.
By enforcing nativelike packing of the A, G, and H helices, the disulfide
resolves local energetic frustration and facilitates transient docking
of the E helix region onto the hydrophobic core but has only a small
effect on the refolding rate. The apomyoglobin folding landscape is
highly rugged, with several energetic bottlenecks that frustrate folding;
relief of any one of the major identified bottlenecks is insufficient
to speed progression to the transition state