title: Probing the non-native H helix translocation in apomyoglobin folding intermediates.
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.
authors.affiliation: Department of Molecular Biology and Skaggs Institute of Chemical Biology, The Scripps Research Institute , 10550 North Torrey Pines Road, La Jolla, California 92037, United States.
authors.forename: Phillip C
authors.initials: PC
authors.lastname: Aoto
authors.forename: Chiaki
authors.initials: C
authors.lastname: Nishimura
authors.forename: H Jane
authors.initials: HJ
authors.lastname: Dyson
authors.forename: Peter E
authors.initials: PE
authors.lastname: Wright
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001059:Apoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003545:Cysteine; D003553:Cystine; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009211:Myoglobin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D011487:Protein Conformation; D017510:Protein Folding; D058849:Protein Refolding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D050661:Sperm Whale; D013816:Thermodynamics
chemical_list: D001059:Apoproteins; D050505:Mutant Proteins; D009211:Myoglobin; D011994:Recombinant Proteins; C010857:apomyoglobin; D003553:Cystine; D003545:Cysteine