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Conformational and Dynamical Characterization of the Molten Globule State of an Apomyoglobin Mutant with an Altered Folding Pathway

Title data

Cavagnero, Silvia ; Nishimura, Chiaki ; Schwarzinger, Stephan ; Dyson, H. Jane ; Wright, Peter E.:
Conformational and Dynamical Characterization of the Molten Globule State of an Apomyoglobin Mutant with an Altered Folding Pathway.
In: Biochemistry. Vol. 40 (2001) Issue 48 . - pp. 14459-14467.
ISSN 1520-4995
DOI: https://doi.org/10.1021/bi011500n

Abstract in another language

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.

Further data

Item Type: Article in a journal
Refereed: Yes
Institutions of the University: Research Institutions
Research Institutions > Research Centres
Research Institutions > Research Centres > Nordbayerisches Zentrum für NMR-Spektroskopie - NMR-Zentrum
Result of work at the UBT: Yes
DDC Subjects: 500 Science > 500 Natural sciences
Date Deposited: 06 Aug 2019 06:45
Last Modified: 06 Aug 2019 06:45
URI: https://eref.uni-bayreuth.de/id/eprint/51779