Abstract

Acid-denatured apomyoglobin (apoMb) contains residual helical structure in the region of the polypeptide which corresponds to the H helix of the folded protein. In order to elucidate the role of this residual secondary structure in the protein folding process and to determine whether residual structure in the denatured state affects either the overall rate of folding or the rate of formation of a burst phase intermediate, we have examined the equilibrium and kinetic folding behavior of a mutant designed to destabilize residual secondary structure in the H helix region. Both Asn132 and Glu136 were changed to Gly (N132G,E136G) to effect this destabilization. Circular dichroism spectra show that the mutant protein contains less helical structure in the acid-denatured state and in the equilibrium intermediate state at pH 4.2 than does the wild-type protein. The CD spectra of the native states of the two proteins are nearly identical. The refolding kinetics for each of the species were measured by stopped-flow CD in the far-UV region and by NMR quench-flow pulse labeling. Under identical conditions, the CD-detected refolding of wild-type and mutant apomyoglobin from the acid-denatured state or from the urea-denatured state occurs at very similar rates following a burst phase that occurs too rapidly to measure by the stopped-flow technique. The urea dependence of the unfolding and refolding rates is consistent with the presence of at least one obligatory on-pathway intermediate in both wild-type and mutant proteins. The kinetic intermediate of the mutant protein is considerably less stable than that of the wild-type protein. Hydrogen exchange pulse labeling experiments indicate that, in contrast to the wild-type protein, the H helix is not stabilized during the burst phase refolding of the mutant but becomes stabilized during the slower phases. While the wild-type and mutant proteins both form compact intermediates, these differ in the content and location of secondary structure. The rate of folding of the AGH subdomain, which takes place prior to the transition state, is substantially slower for the N132G,E136G mutant protein. A strong propensity for spontaneous formation of helical structure in the H helix region is not a prerequisite for efficient folding nor for formation of equilibrium or kinetic intermediates. These observations suggest that while folding of apomyoglobin proceeds through an obligatory intermediate, the precise structure of this intermediate is not critical and its secondary structure may be altered without substantially affecting either the overall refolding kinetics or the integrity of the final folded state. Study holds ProTherm entries: 6215, 6216 Extra Details: the transition is from unfolding to intermediate myoglobin; protein folding; NMR hydrogen-deuterium exchange;,protein folding intermediate

Submission Details

ID: YPvTY2rd

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:31 p.m.

Version: 1

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