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Folding of apomyoglobin: Analysis of transient intermediate structure during refolding using quick hydrogen deuterium exchange and NMR.
Proc Jpn Acad Ser B Phys Biol Sci. 2017; 93(1):10-27.PJ

Abstract

The structures of apomyoglobin folding intermediates have been widely analyzed using physical chemistry methods including fluorescence, circular dichroism, small angle X-ray scattering, NMR, mass spectrometry, and rapid mixing. So far, at least two intermediates (on sub-millisecond- and millisecond-scales) have been demonstrated for apomyoglobin folding. The combination of pH-pulse labeling and NMR is a useful tool for analyzing the kinetic intermediates at the atomic level. Its use has revealed that the latter-phase kinetic intermediate of apomyoglobin (6 ms) was composed of helices A, B, G and H, whereas the equilibrium intermediate, called the pH 4 molten-globule intermediate, was composed mainly of helices A, G and H. The improved strategy for the analysis of the kinetic intermediate was developed to include (1) the dimethyl sulfoxide method, (2) data processing with the various labeling times, and (3) a new in-house mixer. Particularly, the rapid mixing revealed that helices A and G were significantly more protected at the earlier stage (400 µs) of the intermediate (former-phase intermediate) than the other helices. Mutation studies, where each hydrophobic residue was replaced with an alanine in helices A, B, E, F, G and H, indicated that both non-native and native-like structures exist in the latter-phase folding intermediate. The N-terminal part of helix B is a weak point in the intermediate, and the docking of helix E residues to the core of the A, B, G and H helices was interrupted by a premature helix B, resulting in the accumulation of the intermediate composed of helices A, B, G and H. The prediction-based protein engineering produced important mutants: Helix F in a P88K/A90L/S92K/A94L mutant folded in the latter-phase intermediate, although helix F in the wild type does not fold even at the native state. Furthermore, in the L11G/W14G/A70L/G73W mutant, helix A did not fold but helix E did, which is similar to what was observed in the kinetic intermediate of apoleghemoglobin. Thus, this protein engineering resulted in a changed structure for the apomyoglobin folding intermediate.

Authors+Show Affiliations

Faculty of Pharmaceutical Sciences, Teikyo Heisei University.

Pub Type(s)

Journal Article
Review

Language

eng

PubMed ID

28077807

Citation

Nishimura, Chiaki. "Folding of Apomyoglobin: Analysis of Transient Intermediate Structure During Refolding Using Quick Hydrogen Deuterium Exchange and NMR." Proceedings of the Japan Academy. Series B, Physical and Biological Sciences, vol. 93, no. 1, 2017, pp. 10-27.
Nishimura C. Folding of apomyoglobin: Analysis of transient intermediate structure during refolding using quick hydrogen deuterium exchange and NMR. Proc Jpn Acad Ser B Phys Biol Sci. 2017;93(1):10-27.
Nishimura, C. (2017). Folding of apomyoglobin: Analysis of transient intermediate structure during refolding using quick hydrogen deuterium exchange and NMR. Proceedings of the Japan Academy. Series B, Physical and Biological Sciences, 93(1), 10-27. https://doi.org/10.2183/pjab.93.002
Nishimura C. Folding of Apomyoglobin: Analysis of Transient Intermediate Structure During Refolding Using Quick Hydrogen Deuterium Exchange and NMR. Proc Jpn Acad Ser B Phys Biol Sci. 2017;93(1):10-27. PubMed PMID: 28077807.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Folding of apomyoglobin: Analysis of transient intermediate structure during refolding using quick hydrogen deuterium exchange and NMR. A1 - Nishimura,Chiaki, PY - 2017/1/13/entrez PY - 2017/1/13/pubmed PY - 2017/3/28/medline SP - 10 EP - 27 JF - Proceedings of the Japan Academy. Series B, Physical and biological sciences JO - Proc Jpn Acad Ser B Phys Biol Sci VL - 93 IS - 1 N2 - The structures of apomyoglobin folding intermediates have been widely analyzed using physical chemistry methods including fluorescence, circular dichroism, small angle X-ray scattering, NMR, mass spectrometry, and rapid mixing. So far, at least two intermediates (on sub-millisecond- and millisecond-scales) have been demonstrated for apomyoglobin folding. The combination of pH-pulse labeling and NMR is a useful tool for analyzing the kinetic intermediates at the atomic level. Its use has revealed that the latter-phase kinetic intermediate of apomyoglobin (6 ms) was composed of helices A, B, G and H, whereas the equilibrium intermediate, called the pH 4 molten-globule intermediate, was composed mainly of helices A, G and H. The improved strategy for the analysis of the kinetic intermediate was developed to include (1) the dimethyl sulfoxide method, (2) data processing with the various labeling times, and (3) a new in-house mixer. Particularly, the rapid mixing revealed that helices A and G were significantly more protected at the earlier stage (400 µs) of the intermediate (former-phase intermediate) than the other helices. Mutation studies, where each hydrophobic residue was replaced with an alanine in helices A, B, E, F, G and H, indicated that both non-native and native-like structures exist in the latter-phase folding intermediate. The N-terminal part of helix B is a weak point in the intermediate, and the docking of helix E residues to the core of the A, B, G and H helices was interrupted by a premature helix B, resulting in the accumulation of the intermediate composed of helices A, B, G and H. The prediction-based protein engineering produced important mutants: Helix F in a P88K/A90L/S92K/A94L mutant folded in the latter-phase intermediate, although helix F in the wild type does not fold even at the native state. Furthermore, in the L11G/W14G/A70L/G73W mutant, helix A did not fold but helix E did, which is similar to what was observed in the kinetic intermediate of apoleghemoglobin. Thus, this protein engineering resulted in a changed structure for the apomyoglobin folding intermediate. SN - 1349-2896 UR - https://www.unboundmedicine.com/medline/citation/28077807/Folding_of_apomyoglobin:_Analysis_of_transient_intermediate_structure_during_refolding_using_quick_hydrogen_deuterium_exchange_and_NMR_ L2 - https://dx.doi.org/10.2183/pjab.93.002 DB - PRIME DP - Unbound Medicine ER -