General Mechanisms for the Folding and Assembly of Myoglobins and Hemoglobins

Abstract

Mammalian myoglobin has served as the archetype globin for understanding the folding properties of single domain globins with the 3 on 3 helical fold. After removal of heme, the resultant apo-Mb shows a loss of structure in the proximal F helix and adjacent loops, and during acid or GdmCl-induced denaturation, apo-Mb populates at least one intermediate. In contrast, unfolding of holo-Mb appears to be a simple two-state process with little protein concentration dependence but the underlying mechanism is much more complex. The lack of protein concentration dependence implies that heme either interacts with the unfolded polypeptide, self-associates, or both. The observed steepness of the unfolding curves for holo met-Mb requires that the affinity of hemin for the intermediate and completely unfolded states must be at least be 1000 fold weaker than that for the native apo-state, and as a result, unfolding of holo met-Mb is governed primarily by the affinity of the folded native apo-state for hemin. The generality of this conclusion for holo-Mb has been tested in several other monomeric hemoglobins, including the miniglobin from Cerebratulus lacteus and the thermoglobin from Aquifex aeolicus.Human hemoglobin unfolding is even more complex due to association of the α and β subunits into dimers and tetramers. Removal of hemin leads to formation of an apo-α1β1 dimer and its unfolding appears to involve an intermediate whose stability is dependent on protein concentration. This dependence suggests the formation of a dimer intermediate with partially folded subunits still attached to each other through the α1β1 interface. Folding and assembly of holo-Hb is even more complex because there are significant differences in hemin affinity between the α and β subunits, and between tetramers, dimers and monomers.