Effect of H helix destabilizing mutations on the kinetic and equilibrium folding of apomyoglobin

Acid-denatured apomyoglobin (apoMb) contains residual helical structure in the region of the polypeptide which corresponds to the H helix of the folde d protein. In order to elucidate the role of this residual secondary struct ure in the protein folding process and to determine whether residual struct ure in the denatured state affects either the overall rate of folding or th e rate of formation of a burst phase intermediate, we have examined the equ ilibrium and kinetic folding behavior of a mutant designed to destabilize r esidual secondary structure in the H helix region. Both Asn132 and Glu136 w ere changed to Gly (N132G,E136G) to effect this destabilization. Circular d ichroism spectra show that the mutant protein contains less helical structu re 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 state s 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 b y NMR quench-flow pulse labeling. Under identical conditions, the CD-detect ed refolding of wild-type and mutant apomyoglobin from the acid-denatured s tate or from the urea-denatured state occurs at very similar rates followin g a burst phase that occurs too rapidly to measure by the stopped-flow tech nique. The urea dependence of the unfolding and refolding rates is consiste nt 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. Hyd rogen exchange pulse labeling experiments indicate that, in contrast to the wild-type protein, the H helix is not stabilized during the burst phase re folding of the mutant but becomes stabilized during the slower phases. Whil e the wild-type and mutant proteins both form compact intermediates, these differ in the content and location of secondary structure. The rate of fold ing of the AGH subdomain, which takes place prior to the transition state, is substantially slower for the N132G,E136G mutant protein. A strong propen sity for spontaneous formation of helical structure in the H helix region i s 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 stru cture of this intermediate is not critical and its secondary structure may be altered without substantially affecting either the overall refolding kin etics or the integrity of the final folded state. (C) 1999 Academic Press.