Probing the Non-Native H Helix Translocation in Apomyoglobin Folding Intermediates

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• 作者：Phillip C. Aoto ; Chiaki Nishimura ; H. Jane Dyson ; Peter E. Wright
• 刊名：Biochemistry
• 出版年：2014
• 出版时间：June 17, 2014
• 年：2014
• 卷：53
• 期：23
• 页码：3767-3780
• 全文大小：605K
• 年卷期：v.53,no.23(June 17, 2014)
• ISSN：1520-4995

文摘

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. F枚rster 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鈥揕135C 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.