Intrinsic stability of individual alpha helices modulates structure and stability of the apomyoglobin molten globule form.

During acid-induced unfolding of apomyoglobin, a partly folded form is observed at pH values of around four. In this form, the A, G and H helices are folded, while the rest of the molecule, including the B helix, demonstrates little structure. The partly folded form has been described as a molten globule form. To determine the factors that govern the structure and stability of this form, we introduced two helix-stabilizing mutations into the B helix, and tested their effect on the structure and stability of both the native form and the molten globule form. We show that the two Gly-->Ala replacements in the B helix produce altered fluorescence and CD properties of the partly folded intermediate, a result which implies that the B helix has become part of the structured region of the molten globule form. The helix content of a model peptide containing the sequence of the B helix is increased by the G-->A replacements, as is the helix content of the molten globule intermediate, whereas the stability and the helix content of the native protein are not altered. The observed increase in helicity is larger in the folding intermediate than in the model peptide, suggesting that nonspecific interactions, such as the hydrophobic interactions exhibited by the entire polypeptide chain, amplify the effect of intrinsic helix stability. The overall results suggest that the intrinsic stability of each individual helix is a factor in deciding whether or not that helix becomes part of the structured molten globule.