Folding domains as functional tools in allosteric systems: a heme-dependent domain in hemoglobin .beta. subunits

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Abstract
The denaturation by guanidine hydrochloride (Gdn.cntdot.HCl), temperature and pH of [human] Hb .beta. subunits and of the peptides .beta.(1-146), .beta.(56-146) and .beta.(1-55) was studied. The last peptide was insensitive to all of the 3 agents. In the other polypeptides, denaturation by Gdn.cntdot.HCl and temperature showed the presence of several structural domains characterized by different stabilities. Analyses of the data obtained in Gdn.cntdot.HCl indicated the presence in .beta. subunits of an .alpha.-helical domain involving some 40 amino acids whose free energy of denaturation is only 2000 cal. This domain is heme dependent and removal of heme in apo-.beta.-(1-146) abolishes the presence of the domain as a structural entity. Acid denaturation reveals in .beta. subunits, apo-.beta.(1-146) and .beta.(56-146) the presence of buried histidines with very similar characteristics, indicating a similar tertiary structure in the 3 polypeptides. Removal of the heme produces an unfolding of .beta. subunits, involving preferentially the 1-55 portion of the chain. This portion of the polypeptide contributes substantially to the formation of the .alpha.1.beta.2 interface in Hb. The low stability of this domain implies a very small contribution to the stability of the system as a whole. Instead it makes it very sensitive to conformational attitudes of the heme, suggesting a role in the mechanism of ligand binding cooperativity and subunits interactions in Hb.