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Conformational and dynamic characterization of the molten globule state of an apomyoglobin mutant with an altered folding pathway.
Biochemistry. 2001 Dec 04; 40(48):14459-67.B

Abstract

Kinetic and equilibrium studies of apomyoglobin folding pathways and intermediates have provided important insights into the mechanism of protein folding. To investigate the role of intrinsic helical propensities in the apomyoglobin folding process, a mutant has been prepared in which Asn132 and Glu136 have been substituted with glycine to destabilize the H helix. The structure and dynamics of the equilibrium molten globule state formed at pH 4.1 have been examined using NMR spectroscopy. Deviations of backbone (13)C(alpha) and (13)CO chemical shifts from random coil values reveal high populations of helical structure in the A and G helix regions and in part of the B helix. However, the H helix is significantly destabilized compared to the wild-type molten globule. Heteronuclear [(1)H]-(15)N NOEs show that, although the polypeptide backbone in the H helix region is more flexible than in the wild-type protein, its motions are restricted by transient hydrophobic interactions with the molten globule core. Quench flow hydrogen exchange measurements reveal stable helical structure in the A and G helices and part of the B helix in the burst phase kinetic intermediate and confirm that the H helix is largely unstructured. Stabilization of structure in the H helix occurs during the slow folding phases, in synchrony with the C and E helices and the CD region. The kinetic and equilibrium molten globule intermediates formed by N132G/E136G are similar in structure. Although both the wild-type apomyoglobin and the mutant fold via compact helical intermediates, the structures of the intermediates and consequently the detailed folding pathways differ. Apomyoglobin is therefore capable of compensating for mutations by using alternative folding pathways within a common basic framework. Tertiary hydrophobic interactions appear to play an important role in the formation and stabilization of secondary structure in the H helix of the N132G/E136G mutant. These studies provide important insights into the interplay between secondary and tertiary structure formation in protein folding.

Authors+Show Affiliations

Department of Molecular Biology MB-2 and Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Comparative Study
Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

11724558

Citation

Cavagnero, S, et al. "Conformational and Dynamic Characterization of the Molten Globule State of an Apomyoglobin Mutant With an Altered Folding Pathway." Biochemistry, vol. 40, no. 48, 2001, pp. 14459-67.
Cavagnero S, Nishimura C, Schwarzinger S, et al. Conformational and dynamic characterization of the molten globule state of an apomyoglobin mutant with an altered folding pathway. Biochemistry. 2001;40(48):14459-67.
Cavagnero, S., Nishimura, C., Schwarzinger, S., Dyson, H. J., & Wright, P. E. (2001). Conformational and dynamic characterization of the molten globule state of an apomyoglobin mutant with an altered folding pathway. Biochemistry, 40(48), 14459-67.
Cavagnero S, et al. Conformational and Dynamic Characterization of the Molten Globule State of an Apomyoglobin Mutant With an Altered Folding Pathway. Biochemistry. 2001 Dec 4;40(48):14459-67. PubMed PMID: 11724558.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Conformational and dynamic characterization of the molten globule state of an apomyoglobin mutant with an altered folding pathway. AU - Cavagnero,S, AU - Nishimura,C, AU - Schwarzinger,S, AU - Dyson,H J, AU - Wright,P E, PY - 2001/11/29/pubmed PY - 2002/1/10/medline PY - 2001/11/29/entrez SP - 14459 EP - 67 JF - Biochemistry JO - Biochemistry VL - 40 IS - 48 N2 - Kinetic and equilibrium studies of apomyoglobin folding pathways and intermediates have provided important insights into the mechanism of protein folding. To investigate the role of intrinsic helical propensities in the apomyoglobin folding process, a mutant has been prepared in which Asn132 and Glu136 have been substituted with glycine to destabilize the H helix. The structure and dynamics of the equilibrium molten globule state formed at pH 4.1 have been examined using NMR spectroscopy. Deviations of backbone (13)C(alpha) and (13)CO chemical shifts from random coil values reveal high populations of helical structure in the A and G helix regions and in part of the B helix. However, the H helix is significantly destabilized compared to the wild-type molten globule. Heteronuclear [(1)H]-(15)N NOEs show that, although the polypeptide backbone in the H helix region is more flexible than in the wild-type protein, its motions are restricted by transient hydrophobic interactions with the molten globule core. Quench flow hydrogen exchange measurements reveal stable helical structure in the A and G helices and part of the B helix in the burst phase kinetic intermediate and confirm that the H helix is largely unstructured. Stabilization of structure in the H helix occurs during the slow folding phases, in synchrony with the C and E helices and the CD region. The kinetic and equilibrium molten globule intermediates formed by N132G/E136G are similar in structure. Although both the wild-type apomyoglobin and the mutant fold via compact helical intermediates, the structures of the intermediates and consequently the detailed folding pathways differ. Apomyoglobin is therefore capable of compensating for mutations by using alternative folding pathways within a common basic framework. Tertiary hydrophobic interactions appear to play an important role in the formation and stabilization of secondary structure in the H helix of the N132G/E136G mutant. These studies provide important insights into the interplay between secondary and tertiary structure formation in protein folding. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/11724558/Conformational_and_dynamic_characterization_of_the_molten_globule_state_of_an_apomyoglobin_mutant_with_an_altered_folding_pathway_ L2 - https://doi.org/10.1021/bi011500n DB - PRIME DP - Unbound Medicine ER -